Synthesis of 2,4-diaminopyrimidine core-based derivatives and biological evaluation of their anti-tubercular activities

molecules

Article

Synthesis of 2,4-Diaminopyrimidine Core-Based

Derivatives and Biological Evaluation of Their

Anti-Tubercular Activities

Yifan Ouyang ¹, Hao Yang , Peng Zhang , Yu Wang , Sargit Kaur , Xuanli Zhu , Zhe Wang ,

,†

1,†

1

2

1

3,

2,

1,4,

Yutong Sun , Wei Hong *, Yun Fong Ngeow * and Hao Wang

*

1

School of Pharmacy, Ningxia Medical University, Yinchuan 750004, China;

ouyang_pharmacy@163.com (Y.O.); yhao246@163.com (H.Y.); zpzzjzp@163.com (P.Z.);

wangyuxingfu@163.com (Y.W.); jf18700994479@163.com (X.Z.); 13239935127@163.com (Z.W.);

syt931115@163.com (Y.S.)

Department of Pre-Clinical Sciences, Faculty of Medicine and Health Sciences, Universiti Tunku

Abdul Rahman, Sungai Long campus, Kajang 43000, Selangor, Malaysia; sargitk3@gmail.com

School of Chemistry and Chemical Engineering, North Minzu University, Yinchuan 750021, China

KeyLaboratory of Hui Ethnic Medicine Modernization, Ministry of Education, Ningxia Medical University,

Yinchuan 750004, China

2

3

4

*

Correspondence: hongwei136@hotmail.com (W.H.); ngeowyf@utar.edu.my (Y.F.N.); paxhw@yahoo.co.uk

(H.W.); Tel.: +86-135-1920-2157 (W.H.); +60-390-860-288 (Y.F.N.); +86-135-1929-8012 (H.W.)

†

These authors contributed equally to this work.

Received: 4 September 2017; Accepted: 20 September 2017; Published: 22 September 2017

Abstract: Tuberculosis (TB) is a chronic, potentially fatal disease caused by Mycobacterium tuberculosis (Mtb)

The dihyrofolate reductase in Mtb (mt-DHFR) is believed to be an important drug target in anti-TB drug

development. This enzyme contains a glycerol (GOL) binding site, which is assumed to be a useful site

to improve the selectivity towards human dihyrofolate reductase (h-DHFR). There have been previous

attempts to design drugs targeting the GOL binding site, but the designed compounds contain a

hydrophilic group, which may prevent the compounds from crossing the cell wall of Mtb to function

at the whole cell level. In the current study, we designed and synthesized a series of mt-DHFR

inhibitors that contain a 2,4-diaminopyrimidine core with side chains to occupy the glycerol binding

site with proper hydrophilicity for cell entry, and tested their anti-tubercular activity against Mtb

.

H37Ra. Among them, compound 16l showed a good anti-TB activity (MIC = 6.25 µg/mL) with a

signiﬁcant selectivity against vero cells. In the molecular simulations performed to understand the

binding poses of the compounds, it was noticed that only side chains of a certain size can occupy the

glycerol binding site. In summary, the novel synthesized compounds with appropriate side chains,

hydrophobicity and selectivity could be important lead compounds for future optimization towards

the development of future anti-TB drugs that can be used as monotherapy or in combination with

other anti-TB drugs or antibiotics. These compounds can also provide much information for further

studies on mt-DHFR. However, the enzyme target of the compounds still needs to be conﬁrmed by

pure mt-DHFR binding assays.

Keywords: anti-tuberculosis; 2,4-diaminopyrimidine derivatives; synthesis

1

. Introduction

There is an urgent need to develop new drugs for the treatment of tuberculosis (TB), a chronic

disabling infection caused by Mycobacterium tuberculosis (Mtb). This pathogen has developed

resistance to standard ﬁrst- and second-line anti-TB drugs, leaving very few options for effective

therapy. para-Aminosalicylic acid (PAS) is a key anti-TB drug that has been in use for over 60 years.

Molecules 2017, 22, 1592; doi:10.3390/molecules22101592

www.mdpi.com/journal/molecules

Molecules 2017, 22, 1592

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Its anti-mycobacterial mechanism was not clearly understood until recently, when it was reported to be

the pro-drug of an inhibitor of the Mtb dihyrofolate reductase (mt-DHFR) [ ]. mt-DHFR catalyzes the

1

reduction of dihydrofolate to tetrahydrolate in the folate metabolic pathway that leads to the synthesis

of purines, pyrimidines and other proteins. Inhibition of the enzyme would cause cell death via the

inhibition of DNA synthesis. This association of PAS with mt-DHFR inhibition encouraged scientists

to focus once again on mt-DHFR as a potential target for anti-TB drugs.

According to their chemical structures, DHFR inhibitors can be divided into “classical” and

“

non-classical” types [

methotrexate (Figure 1), which is a commonly used anticancer drug [

non-classical inhibitors is 2,4-diaminopyrimidine, as in trimethoprim (Figure 1), an anti-bacterial

drug [ ]. The crystal structures of mt-DHFR (PDB ID: 1DF7, Figure 2) and human DHFR

h-DHFR, PDB ID: 1OHJ), show a glycerol (GOL) binding site in mt-DHFR that does not exist in

2

,

3

]. The structures of classical inhibitors are similar to that of folate, as in

4–6]. The core structure of

7–9

(

h-DHFR. To take advantage of this difference, Threadgill evaluated a group of compounds containing

glycerol-like side chains, among which one compound, El-7a (Figure 1) showed notable selectivity

for mt-DHFR inhibition over h-DHFR [10]. However, this evaluation was conducted using TB5

Saccharomyces cerevisiae carrying mt-DHFR and h-DHFR genes, therefore, there is no direct evidence

to show that El-7a can inhibit the growth of Mtb. Furthermore, the inhibition of Mtb may require

an appropriate lipophilicity in the compound [11]. Even if El-7a were able to selectively inhibit the

mt-DHFR, its low hydrophobicity may prevent it from passing through the Mtb cell wall. Hence,

we designed and synthesized a series of compounds containing more hydrophobic groups on the

6-position of 2,4-diaminopyrimidine to evaluate the ability of these compounds to inhibit Mtb

cells directly.

Figure 1. The structures of “classical” and “non-classical” DHFR inhibitors.

Figure 2. Left: the binding sites of methotrexate (MTX) and glycerol (GOL) in mt-DHFR, in which

MTX is represented as a sticks model, GOL as a ball-stick model, and protein as a molecular surface;

Right: the designed molecule is predicted to be able to occupy the GOL binding site, in which the

molecule is represented as sticks and protein as a molecular surface.

Molecules 2017, 22, 1592

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2

. Results and Discussion

2

.1. Chemistry

The synthesis of the designed compounds 10a-q or 11a-q was carried out in five

steps—chlorination, nucleophilic substitution, iodination, Suzuki reaction and deprotection—according

to our patented method [12], with 2,4-diamino-6-hydroxypyrimidine ( ) as the starting material

Scheme 1). Initially, 2,4-diamino-6-chloropyrimidine ( ) was generated from by treatment with

1

(

2

1

phosphorus oxychloride. After the reaction was quenched with ice water, the solution was hydrolyzed

◦

at 90 C to obtain a good yield (85%) of the target intermediate [13]. This procedure yields pure

without the need for chromatography.

2

NH2

OH

O

I

N

O

H2N

N

O

H2N

N

O

NH₂

NH2

3

O

5

O

^P^O^C^l3

N

NaH, DMSO

NIS

H N

2

N

OH

H2N

N

Cl

NH2

N

NH₂

NaH, DMSO

OH

CH CN

3

I

1

2

N

H N

2

O

H N

2

N

O

4

6

NH₂

N

R¹

NH₂

N

R¹

N

H N

2

O

H N

2

O

R¹

HO

B

O

HO

OH

8

a-q

7

0.25M H2SO4

10a-q

(PPh3)4Pd, K2CO3,

EtOH/Tol/H2O

or C26H44Cl2FeP2Pd,

K2CO3, CH3CN/H2O

NH2

N

R¹

NH2

_R1

N

H2N

O

H N

2

N

O

HO OH

O

9

a-q

11a-q

8

a, 9a, 10a, 11a, R¹= 4-Cl;

_m_,₉_m_,₁₀_m_,₁₁_m_,_R1 = 3-CH3-5-CH3-4-SO2NHOCH3;

8

1

b, 9b, 10b, 11b, R = 4-F;

O

c, 9c, 10c, 11c, R¹= 3,4-Cl;

1

d, 9d, 10d, 11d, R = 3-Cl-4-F;

_n_,₉_n_,₁₀_n_,₁₁_n_,_R1

4

8

=

N

1

e, 9e, 10e, 11e, R = 4-CF3;

1

O

f, 9f, 10f, 11f, R = 3-CF₃;

g, 9g, 10g, 11g, R¹= 4-OCF₃;

O

1

h, 9h, 10h, 11h, R = 3-OCF₃;

i, 9i, 10i, 11i, R¹= 4-CN

8o, 9o, 10o, 11o, R¹

=

4

N

H

OCF3

_j_,₉_j_,₁₀_j_,₁₁_j_,_R1 = 4-OCH ;

3

1

8p, 9p, 10p, 11p, R¹= 4-COOCH3;

8q, 9q, 10q, 11q, R¹= 3-COOCH₃;

k, 9k, 10k, 11k, R = -3,4,5-OCH₃;

l, 9l, 10l, 11l, R¹= 3-CH OCH CF ;

2

3

Scheme 1. Synthesis of 2,4-diamino-5-aryl-6-substituted pyrimidine derivatives 10a-q and 11a-q.

The treatment of (S)-2,3-isopropylidene glycerol or (R)-2,3-isopropylidene glycerol with sodium

hydride in dry DMSO generated the corresponding nucleophile, which was then reacted with to give

,4-diamino-6-substituted pyrimidines or in good yield (77%) [14]. Subsequently, the 5-positions

of or were iodinated with N-iodosuccinimide in dry acetonitrile to produce the precursor

,4-diamino-5-iodo-6-substituted pyrimidine derivatives or in 96–98% yields [15]. Initially, we used

a previously synthesized 2,4-diamino-5-bromo-6-substituted pyrimidine derivative as the starting

2

3

4

3

4

2

5

6

Molecules 2017, 22, 1592

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material in the model reaction of the Suzuki reactions in order to introduce a substituted aryl group in

the 5-position of the 2,4-diaminopyrimidine core. We tried several conditions, such as reacting with

phenylboronic acid, 4-chlorophenylboronic acid and 4-methoxycarbonylphenylboronic acid, different

catalysts (Pd(PPh ) [16], Pd(dbpf)Cl ) [17], different bases (K CO , K HPO ), different solvents

3

4

2

3

2

4

(

EtOH/Toluene/H O, THF/H O, CH CN/H O, Dimethoxyethane/H O), different temperatures

2 2 3 2 2

70, 80, 90, 120 C), and different heating modes (microwave or oil bath). Unfortunately, all these

◦

reactions failed. Subsequently, we realized that the iodide replacement in the 5-position of the

,4-diaminopyrimidine core in the Suzuki reaction might be easier than with a bromide. Hence,

the iodides or were allowed to react with substituted phenylboronic acids under Suzuki reaction

conditions (Pd(PPh ) as catalyst, K CO as base, and EtOH/toluene/H O as solvent). However,

2

5

6

7

3

4

2

3

2

the products 8a-o or 9a-o were only obtained in 52–78% yields. Moreover, the presence of an electron

donating (OCH ) or electron withdrawing (F, CF , OCF , ester, amide) group on the substituted

3

phenylboronic acid

7 did not show high effect of the reaction yields. However, when the products

8

p-q or 9p-q were synthesized by reacting

5

or

6

with 4- or 3-methoxycarbonylphenylboronic acids 7p

1

or 7q in EtOH/toluene/H O, the transesteriﬁcation product was detected in the H-NMR sprectrum.

2

Hence, different Suzuki reaction conditions were chosen, in which

5 or 6 was reacted with 7p or 7q

with Pd(dbpf)Cl as catalyst, K CO as base and CH CN/H O as solvent, to give 8p-q or 9p-q with

2

3

2

6

3–78% yields. Subsequently, the compounds 8a-q or 9a-q were deprotected in 0.25 M H SO solution

2 4

to generate the target compounds 10a-q or 11a-q in 68–95% yields.

Subsequently 2,4-diamino-5-aryl-6-substituted pyrimidine derivatives 16a-p were synthesized

from 2,4-diamino-6-chloropyrimidine ( , Scheme 2) following our patented approach [18].

The treatment of substituted methanols 12a-d with sodium hydride in dry DMSO or THF generated the

corresponding nucleophiles, which were reacted with to give 2,4-diamino-6-substituted pyrimidines

3a-d in moderate to good yields (61–79%) [14]. Subsequently, the 5-positions of 13a-d were iodinated

with N-iodosuccinimide in dry acetonitrile to produce the precursor 2,4-diamino-5-iodo-6-substituted

pyrimidine derivatives 14a-d 15], which were used in subsequent Suzuki reactions to generate the

2

1

[

target compounds 16a-p in good yields (70–92%) [17].

Scheme 2. Synthesis of 2,4-diamino-5-aryl-6-substituted pyrimidine derivatives 16a-p.

A series of reactions in which iodides 14a-d were used as staring material, was investigated

as shown in Table 1. The iodides 14a-d were reacted with substituted phenylboronic acid 15 in the

◦

presence of Pd(dbpf)Cl and K CO in EtOH/toluene/H O at 90 C for 24 h (Table 1, Entries 1–14

)

2

3

2

◦

or in the presence of Pd(dbpf)Cl₂and K CO₃in THF/H O at 70 C for 20 h in a sealed

2

tube (Table 1, Entries 15, 16) to generate the desired compounds 16a-p with moderate to good

Molecules 2017, 22, 1592

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yields (51–99%). For the iodide 14a, treatment with different substituted phenylboronic acids

(Table 1, Entries 1–5) resulted in similar yields. For the iodide 14b, reaction with 15

1

5

bearing a 3-triﬂoromethoxy group resulted in higher yields (Table 1, Entry 7) than when 15

bore a 4-triﬂoromethoxy group (Table 1, Entry 6). For the iodide 14c, treatment with 15

substituted with a 3-triﬂoromethoxyanilinocarbonyl group or 3-(2,2,2-triﬂoroethoxymethyl) group

(Table 1, Entries 10, 12) resulted in higher yields than with 4-triﬂoromethoxy, 3-triﬂoromethoxy

and 3,5-dimethyl-4-(N-methoxyaminosulfonyl) groups substituted on 15 (Table 1, Entries 8, 9, 11).

Especially for the iodide 14d, reaction with 15 bearing a 4-triﬂoromethoxy or 3-triﬂoromethoxy group

(Table 1, Entries 13, 14) resulted in much higher yields than with 15 bearing a 4-methoxycarbonyl

or 3-methoxycarbonyl group (Table 1, Entries 15, 16). The presumption is that the ester group on

this Suzuki reaction could be affected by transesteriﬁcation in the solvent (EtOH/toluene/H O),

2

1

which could be detected in H-NMR. Thus, in these reactions, we chose THF/H O as the solvent to

2

avoid the transesteriﬁcation.

Table 1.

Synthesis of 2,4-diamino-5-aryl-6-substituted pyrimidine derivatives 16a-p via

Suzuki reactions.

Entry

R²

R³

Product

16a ^a

Yield ^c(%)

Clog P ^d

1

2

3

-CH OCH (14a)

4-OCF₃

3-OCF₃

3-CH OCH CF

74

70

2.99

2.29

2

3

a

16b

2

3

a

16c

2

3

2

3

-CH -5-CH -4-

3 3

16d ^a

4

-CH OCH (14a)

73

1.19

2

3

SO NHOCH

2

3

5

-CH OCH (14a)

16e ^a

16f ^a

70

3.59

2

3

6

7

-CH CH OCH (14b)

4-OCF₃

3-OCF₃

75

94

3.37

2

3

a

-CH CH OCH (14b)

16g

2

3

8

9

4-OCF₃

3-OCF₃

16h ^a

16i ^a

16j ^a

16k ^a

87

73

91

72

2.81

2.11

1.01

(

14c)

1

0

3-CH OCH CF

2 2 3

3

-CH -5-CH -4-

3 3

SO2NHOCH3

1

2

3

4

5

6

16l ^a

16m ^a

16n ^a

16o ^b

16p ^b

99

91

68

51

3.42

3.56

2.39

(

14c)

1

4-OCF₃

3-OCF₃

N

(14d)

N

(14d)

4-COOCH₃

3-COOCH₃

Reaction Conditions: ^aPd(dbpf)Cl

0.02 mmol) and K CO

(0.02 mmol) and K

CO

in EtOH/toluene/H

O at 90 C for 24 h; Pd(dbpf)Cl

◦

b

2

3

2

◦

c

d

(

2

3

in THF/H

2

O at 70 C for 20 h in sealed tube; Isolated yields; Calculated using

ChemBioDraw (PerkinElmer, Waltham, MA, USA) 12.0.

Molecules 2017, 22, 1592

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2

.2. Determination of In Vitro Anti-Tubercular Activity

2

Based on the different R substituents on the 2,4-diamino-5-aryl-6-substituted pyrimidine

2

derivatives, the compounds can be divided into four types: (1) the R substituents bearing hydroxy

2

groups (10a-q

,

11a-q); (2) the R substituents bearing alkoxy groups (16a-g); (3) the R substituents

2

bearing thiazole groups (16h-l); (4) the R substituents bearing phenyl substituted triazole groups

(

16m-p). Only compounds containing the thiazole group act as Mtb inhibitors. Among this group of

compounds, ﬁve compounds (16h-l) showed potentially useful inhibitory effects, with 16l showing the

lowest MIC (6.25 g/mL or 12.45 M) and MBC (12.5 g/mL) (Table 2). In order to see the selectivity of

6l against mammalian cells, the MTT assay was performed on vero cells, and the IC₅₀on cells viability

µ

1

was found to be around 50.22 µM. The selectivity ratio of 16l on H37Ra vs vero cells is around 4-fold.

Table 2. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC)

of compounds 16h-l showing anti-tubercular activity.

Compound

6h

R²

R³

MIC/MBC (µg/mL)

1

4-OCF₃

25/25

1

6i

6j

3-OCF₃

50/50

25/25

1

3-CH OCH CF

2 2 3

3

-CH -5-CH -4-

3 3

1

6k

100/100

SO NHOCH

2

3

1

6l

6.25/12.5

Rifampicin

0.313/0.313

2

.3. Molecular Docking and Simulation

Through the structural analysis of the compounds, the Clog P of El-7a was noticed to be

which showed the compound El-7a to be very hydrophilic, and led us to assume it would not be

able to cross the Mtb cell wall. This assumption was indirectly conﬁrmed by the observation that

−

0.17,

the hydrophilic compounds 10a-q and 11a-q (analogs of El-7a, with Clog P around

−

1 to 2), could

not inhibit the growth of Mtb. Based on the above assumption, more hydrophobic compounds were

analyzed by using molecular docking and molecular dynamic simulations, and based on the size of the

substituents on the 6-position of 2,4-diaminopyrimidine, they were divided into three groups, which

are large side chain groups (compounds 16m-p), medium side chain groups (compounds 16h-l) and

small side chain groups (compounds 16a-g). With molecular docking, it was noticed that the large side

chain group, which contains the 1-benzyl-1H-1,2,3-triazole-4-methoxy group on the 6-position, cannot

ﬁt into the GOL binding site (Figure 3a), and this could be the reason why this group of compounds did

not show any inhibition effects on Mtb. Although the small side chain group derivatives (compounds

1

6a-g), which contain the methoxyethoxy or methoxypropoxy group on the 6-positions, can ﬁt into the

GOL binding site (Figure 3b,c), they cannot form strong interactions or fully ﬁll the GOL binding site.

Molecules 2017, 22, 1592

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(

a)

(b)

(

c)

Figure 3. (a) Compound 16m with large side chain group and the binding sites of glycerol (GOL) in

mt-DHFR, in which compound 16m is represented as a sticks model, GOL as a dotted line model and

protein as a molecular surface; ( ) the small side chain group of compound 16f can ﬁt into the GOL

b,c

binding site, in which the molecule is represented as sticks and protein as a molecular surface.

The molecular docking showed that the medium side chain group (compounds 16h-l),

which contain a (thiazol-5-yl)methoxy on the 6-position, can ﬁt into the GOL binding site properly,

and a molecular dynamics simulation was performed to understand the binding of compound 16l

to mt-DHFR. During 100 ns simulations, 16l was stable in the binding site, and the side chain of

compounds 16h-l occupied the GOL binding site along the full simulation (Figure 2). The free energy

calculation showed that the binding free energy was

−

3.47 Kcal/mol (Table 3), which indicated that

1

6l can bind with mt-DHFR tightly. The free energy contributions of each residue was calculated, and

contributions greater than

−

0.5 Kcal/mol were recorded (Ile20, Arg23, Phe31, Leu50, Pro51 and Val54)

(Figure 4 Left). Most of these residues showed a strong VDW interaction, except Phe31 and Arg23

which formed a H-bond with 16l. Arg23 could also form strong interactions with the triﬂuoromethoxy

group (Figure 4 Right).

Table 3. Binding free energies (Kcal/mol) of compound 16l in mt-DHFR.

Simulations ∆E_v_d_w

42.37

0.14

∆E_e_l_e

∆G_p_b

∆Gnp

∆Ggas

∆G_s_o_l_v

∆G_m_m_p_b_s_aT∆S

∆G_b_i_n_d_i_n_g

−3.47

−

−18.16

± 0.24

37.55 ±

−5.62

± 0.01

−60.53

± 0.30

31.93 ±

−28.60

± 0.18

−25.13

± 0.73

1

6l

±

0.33

0.32

Molecules 2017, 22, 1592

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0

-

0.5

-

1

1.5

-

2

Figure 4. Left: The residues whose binding free energy contributions are greater than

Right: The interactions between key residues and compound 16l.

−

0.5 Kcal/mol;

Therefore, through the molecular docking and molecular dynamic simulations, we believe that

the compounds containing the (thiazol-5-yl)methoxy side chain (medium size group), can fully occupy

the GOL binding site, and have reasonable properties. Therefore, such compounds could be used as

the lead compounds for further anti-TB drug discovery studies.

3

. Materials and Methods

3

.1. General Information

All reagents and solvents were purchased from the suppliers and used directly in the experiments.

THF was dried by distillation ver sodium benzophenone. TLC was carried out using silica gel 60

pre-coated aluminium plates (0.20 mm thickness) from Macherey-Nagel (Darmstadt, Germany) with

visualisation by UV light (254 nm). Flash chromatography was performed on silica gel (particle size

4

0–63

µ

m). IR spectra were recorded on a Tensor 27 spectrometer (Bruker, Ettlingen, Germany) using

1

KBr discs. H-NMR spectra were obtained from an AVANCE III 400 spectrometer (Bruker, Fällanden

Switzerland). The chemical shifts, given as

1

δ

values, were quoted in parts per million (ppm); H-NMR

chemical shifts were measured relative to internal tetramethylsilane; Apparent coupling constants

absolute values), J, were measured in Hertz and multiplicities quoted as singlet (s), doublet (d),

triplet (t), quartet (q) or combinations thereof as appropriate. Mass spectra were obtained from an

545 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA). Melting points

(

6

were determined using a WRS-1B melting point measurement instrument (Shanghai, China) and

were uncorrected.

3

.2. ChemistryIt

,4-Diamino-6-chloropyrimidine (

2

). 2,4-Diamino-6-hydroxypyrimidine (

1

) (1.00 g, 7.93 mmol) was

◦

added to POCl (9 mL), and stirred at 97 C for 17 h. The reaction solution was added to ice water

slowly, and then stirred at 90 C for 1 h. The pH of this solution was adjusted to 8 with NaOH,

3

◦

and then it was extracted with EtOAC (150 mL

×

3). The combined organic layers were dried with

◦

Na SO , ﬁltered and concentrated to give white solid 0.97 g, yield 85%. m.p. 200.2–200.4 C; IR

2

4

−

1

(

KBr): υmax/cm 3449 (NH), 3327 (NH), 1642 (C=N), 1581 (C=C), 1551 (C=C), 795 (C-Cl); H-NMR

+

(

DMSO-d₆)

δ

6.57 (s, 2H, NH ), 6.31 (s, 2H, NH ), 5.69 (s, 1H, Ar-H); ES-MS 145.0 (M + H) ; HRMS

2

+

Calcd. for C H ClN O 145.0281, found 145.0276.

16

20

4

3

Molecules 2017, 22, 1592

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General Procedure for the Synthesis of Compounds 3 and 4

Under argon, to a solution of (S)-2,3-isopropylideneglycerol or (R)-2,3-isopropylideneglycerol

0

.50 mL (4.0 mmol) in dry DMSO (5 mL) was added NaH 0.20 g (60%, 5.0 mmol) and stirred at room

temperature for 1 h. 2,4-Diamino-6-chloropyrimidine (

at 90 C for 8 h. The reaction solution was quenched with sat NH Cl (20 mL) and extracted with

2

, 0.29 g, 2.0 mmol) was added and stirred

◦

4

EtOAc (30 mL

×

3), and the combined organic layers dried with Na SO , ﬁltered and concentrated.

2 4

The residue was puriﬁed by column chromatography on silica gel using CH Cl /CH OH (50:1, v/v)

2

3

as the eluting solvent to give compounds 3 or 4.

(

R)-2,4-Diamino-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (3). White solid 0.37 g, yield 77%;

◦

−1

m.p. 89.7–91.0 C; IR: υmax/cm 3474 (NH), 3452 (NH), 3373 (NH), 3359 (NH), 1668 (C=N),

1

627 (C=N), 1593 (C=C), 1200 (C-O-C), 1157 (C-O-C), 1082 (C-O-C), 1052 (C-O-C); H-NMR (CDCl )

3

δ

5.28 (s, 1H, Ar-H), 4.71 (s, 2H, NH ), 4.53 (s, 2H, NH ), 4.41 (q, J = 6.0, 1H, CH), 4.25 (d, J = 5.6, 2H,

2 2

OCH ), 4.11 (dd, J = 8.2, J = 6.4, 1H, OCH ), 3.81 (dd, J = 8.4, J = 6.4, 1H, OCH ), 1.44 (s, 3H, CH ),

2

1

2

1

2

3

+

1

.38 (s, 3H, CH ); ES-MS 241.1 (M + H) ; HRMS Calcd. for C H ClN O 241.1301, found 241.1303.

3 16 20 4 3

(

S)-2,4-Diamino-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (4). White solid 10.32 g, yield 77%;

◦

−1

m.p. 87.6–88.9 C; IR: υmax/cm 3474 (NH), 3451 (NH), 3373 (NH), 3358 (NH), 1670 (C=N),

1

626 (C=N), 1592 (C=C), 1199 (C-O-C), 1159 (C-O-C), 1085 (C-O-C), 1054 (C-O-C); H-NMR (CDCl )

3

δ

5.29 (s, 1H, Ar-H), 4.67 (s, 2H, NH ), 4.50(s, 2H, NH ), 4.41 (q, J = 6.0, 1H, CH), 4.29–4.22 (m, 2H,

2 2

OCH ), 4.11 (dd, J = 8.8, J = 6.4, 1H, OCH ), 3.82 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.45 (s, 3H, CH ),

2

1

2

1

2

3

+

1

.38 (s, 3H, CH ); ES-MS 241.1 (M + H) ; HRMS Calcd. for C H ClN O 241.1301, found 241.1306.

3 16 20 4 3

General Procedure for the Synthesis of Compounds 5 and 6

Under argon, to a solution of or (7.18 g, 29.88 mmol) in dry CH CN (100 mL) was added

N-iodosuccinimide 10.09 g (44.83 mmol) and stirred at room temperature for 1 h. The reaction

solution was diluted with EtOAc (500 mL), washed by 5% NaHSO (500 mL), NaHCO (500 mL)

3

4

3

and H O (500 mL), and dried with Na SO , ﬁltered and concentrated. The residue was puriﬁed by

2

4

column chromatography on silica gel using CH Cl /CH OH (100:1, v/v) as the eluting solvent to give

2

3

compounds 5 or 6.

(

9

1

R)-2,4-Diamino-5-iodo-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (5). White solid 13.42 g, yield

◦

−1

8%; m.p. 135.5–136.8 C; IR: υmax/cm 3464 (NH), 3402 (NH), 3356 (NH), 1649 (C=N), 1627 (C=N),

1

550 (C=C), 1200 (C-O-C), 1157 (C-O-C), 1082 (C-O-C), 1052 (C-O-C), 476 (C-I); H-NMR (CDCl )

δ

3

5

.06 (s, 2H, NH ), 4.69 (s, 2H, NH ), 4.44–4.37 (m, 2H, OCH and CH), 4.30–4.25 (m, 1H, OCH ), 4.12

2

(

dd, J = 8.4, J = 6.4, 1H, OCH ), 3.96 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.47 (s, 3H, CH ), 1.39 (s, 3H,

1 2 2 1 2 2 3

+

CH ); ES-MS 367.0 (M + H) ; HRMS Calcd. for C H ClN O 367.0267, found 367.0265.

3

16 20

4

3

(

9

S)-2,4-Diamino-5-iodo-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (

6%; m.p. 135.4–136.7 C; IR: υmax/cm 3465 (NH), 3401 (NH), 3360 (NH), 3310 (NH), 1650 (C=N),

6

). White solid 10.72 g, yield

◦

−1

1

623 (C=N), 1549 (C=C), 1200 (C-O-C), 1157 (C-O-C), 1133 (C-O-C), 1096 (C-O-C), 1071 (C-O-C), 1045

1

(C-O-C), 478 (C-I); H-NMR (CDCl )

δ

5.05 (s, 2H, NH ), 4.68 (s, 2H, NH ), 4.37–4.34 (m, 2H, OCH

2

3

2

and CH), 4.30–4.25 (m, 1H, CH ), 4.12 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.96 (dd, J = 8.4, J = 6.0, 1H,

2

1

2

1

2

+

OCH ), 1.47 (s, 3H, CH ), 1.39 (s, 3H, CH ); ES-MS 367.0 (M + H) ; HRMS Calcd. for C H ClN O

3

2

3

16 20

4

3

67.0267, found 367.0268.

General Procedure for the Synthesis of Compounds 8a-q and 9a-q

(

A) Under argon, to a mixed solution of EtOH/toluene (1:2, 60 mL) was added compound

5

or

6

−

4

(

2.73 mmol), substituted phenylboronic acid (

K CO (3 M, 3.00–5.50 mL) consecutively and then stirred at 90 C for 1–2 h. The reaction solution

7

) (3.00–5.46 mmol), Pd(PPh ) (1.38

×

10 mmol) and

3

4

◦

2

3

was extracted with EtOAc (30 mL

×

3), and the combined organic layers were washed by H O and

2

Molecules 2017, 22, 1592

10 of 27

dried with Na SO , ﬁltered and concentrated. The residue was puriﬁed by column chromatography

2

4

on silica gel using CH Cl /CH OH (80:1, v/v) as the eluting solvent to the desired compounds.

2

3

(

B) In a pressure tube, to a mixed solution of CH CN/H O (1:1, 40 mL) was added compound 5 or

3 2

−

4

6

(2.73 mmol), substituted phenylboronic acid 7p or 7q (4.10 mmol), Pd(dbpf)Cl (2.73 × 10 mmol

)

2

◦

and K CO (4.10 mmol) consecutively and then stirred at 60 C for 8 h. The reaction solution was

2

3

extracted with EtOAc (30 mL

×

3), and the combined organic layers were washed by H O and dried

2

with Na SO , ﬁltered and concentrated. The residue was puriﬁed by column chromatography on silica

2

4

gel using CH Cl /CH OH (60:1, v/v) as the eluting solvent to the desired compounds.

2

3

(

R)-2,4-Diamino-5-(4-chlorophenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (8a). Method A.

◦

−1

White solid 0.64 g, yield 67%; m.p. 171.2–172.8 C; IR: υmax/cm 3475 (NH), 3454 (NH), 3305 (NH),

3

315 (NH), 1649 (C=N), 1620 (C=N), 1592 (C=C), 1549 (C=C), 1479 (C=C), 1160 (C-O-C), 1144 (C-O-C),

1

1089 (C-O-C), 794 (C-Cl); H-NMR (CDCl )

δ

7.36 (d, J = 8.4, 2H, Ar-H), 7.25 (d, J = 8.0, 2H, Ar-H), 4.69

3

(

s, 2H, NH ), 4.52 (s, 2H, NH ), 4.36 (dd, J = 10.8, J = 4.4, 1H, OCH ), 4.31–4.26 (m, 1H, CH), 4.20

2 2 1 2 2

dd, J = 10.8, J = 5.6, 1H, OCH ), 3.97 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.72 (dd, J = 8.4, J = 6.0, 1H,

1

2

1

2

1

2

+

OCH ), 1.32 (s, 3H, CH ), 1.28 (s, 3H, CH ); ES-MS 351.1 (M + H) ; HRMS Calcd. for C H ClN O

3

2

3

16 20

4

3

51.1224, found 351.1222.

(

R)-2,4-Diamino-5-(4-ﬂuorophenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (8b). Method A.

◦

−1

White solid 0.61 g, yield 74%; m.p. 162.3–163.0 C; IR: υmax/cm 3526 (NH), 3483 (NH), 3369 (NH),

3

312 (NH), 1647 (C=N), 1626 (C=N), 1593 (C=C), 1558 (C=C), 1510 (C=C), 1485(C=C), 1383 (C-F), 1160

1

(C-O-C), 1128 (C-O-C), 1105 (C-O-C), 1090 (C-O-C); H-NMR (CDCl )

δ

7.27 (dd, J = 8.8, J = 5.6,

3

1

2

H, Ar-H), 7.08 (t, J = 8.8, 2H, Ar-H), 4.69 (s, 2H, NH ), 4.52 (s, 2H, NH ), 4.36 (dd, J = 10.8, J = 4.0,

2 2 1 2

1

H, OCH ), 4.31–4.26 (m, 1H, CH), 4.20 (dd, J = 10.8, J = 5.6, 1H, OCH ), 3.96 (dd, J = 8.4, J = 6.4,

2 1 2 2 1 2

H, OCH ), 3.71 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ), 1.27 (s, 3H, CH ); ES-MS 335.2

2

1

2

3

+

(M + H) ; HRMS Calcd. for C H FN O 335.1519, found 335.1515.

16 20 4 3

(

R)-2,4-Diamino-5-(3,4-dichlorophenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (8c). Method A.

◦

−1

White solid 0.55 g, yield 52%; m.p. 164.1–165.9 C; IR: υmax/cm 3487 (NH), 3378 (NH), 3316 (NH),

1

651 (C=N), 1624 (C=N), 1600 (C=C), 1556 (C=C), 1489 (C=C), 1154 (C-O-C), 1134 (C-O-C), 1191 (C-O-C),

1

073 (C-O-C), 795 (C-Cl); H-NMR (CDCl )

δ 7.45 (d, J = 8.0, 1H, Ar-H), 7.44 (d, J = 2.0, 1H, Ar-H),

3

7

.17 (dd, J = 8.0, J = 2.0, 1H, Ar-H), 4.73 (s, 2H, NH ), 4.56 (s, 2H, NH ), 4.35 (dd, J = 10.4, J = 4.4,

1 2 2 2 1 2

1

H, OCH ), 4.33–4.27 (m, 1H, CH), 4.22 (dd, J =10.4 , J = 5.2, 1H, OCH ), 3.99 (dd, J = 8.4, J = 6.0,

2 1 2 2 1 2

H, OCH ), 3.73 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.33 (s, 3H, CH ), 1.30 (s, 3H, CH ); ES-MS 385.1

2

1

2

3

+

(M + H) ; HRMS Calcd. for C H Cl N O 385.0834, found 385.0830.

16 19 2 4 3

(

R)-2,4-Diamino-5-(3-chloro-4-ﬂuorophenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine

(

8d).

◦

−1

Method A. White solid 0.64 g, yield 64%; m.p. 149.1–150.1 C; IR: υmax/cm 3484 (NH), 3382 (NH),

3

320 (NH), 1650 (C=N), 1621(C=N), 1594 (C=C), 1553 (C=C), 1502 (C=C), 1372 (C-F), 1160 (C-O-C),

1

7

4

3

092 (C-O-C), 1065 (C-O-C), 797 (C-Cl); H-NMR (CDCl )

.20–7.14 (m, 2H, Ar-H), 4.71 (s, 2H, NH ), 4.53 (s, 2H, NH ), 4.35 (dd, J = 10.6, J = 4.2, 1H, OCH ),

.32–4.27 (m, 1H, CH), 4.22 (dd, J = 10.6, J = 5.4, 1H, OCH ), 3.98 (dd, J = 8.4, J = 6.0, 1H, OCH ),

.72 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.33 (s, 3H, CH ), 1.29 (s, 3H, CH ); ES-MS 369.1 (M + H) ;

δ

7.37 (dd, J = 7.2, J = 1.6, 1H, Ar-H),

3

1

2

1

2

1

2

1

2

+

1

2

3

+

HRMS Calcd. for C H ClFN O , 369.1130, found 369.1128.

16

19

4

3

(

R)-2,4-Diamino-5-(4-triﬂuoromethylphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine

(

8e).

◦

−1

Method A. Light yellow solid 0.72 g, yield 69%; m.p. 126.6–127.2 C; IR: υmax/cm 3530 (NH), 3511

(

NH), 3444 (NH), 3420 (NH), 1649 (C=N), 1609 (C=N), 1593 (C=C), 1558 (C=C), 1491 (C=C), 1171

1

C-O-), 1128 (C-O-C), 1094 (C-O-C), 1072 (C-O-C), 1046 (C-O-C); H-NMR (CDCl )

δ

7.64 (d, J = 8.2,

3

2

H, Ar-H), 7.46 (d, J = 8.2, 2H Ar-H), 4.73 (s, 2H, NH ), 4.56 (s, 2H, NH ), 4.37 (dd, J = 11.2, J = 4.2,

2 2 1 2

1

H, OCH ), 4.32–4.27 (m, 1H, CH), 4.22 (dd, J = 11.2, J = 5.6, 1H, OCH ), 3.97 (dd, J = 8.4, J = 6.0,

2 1 2 2 1 2

H, OCH ), 3.71 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ), 1.24 (s, 3H, CH ); ES-MS 385.1

2

1

2

3

+

(M + H) ; HRMS Calcd. for C H F N O 385.1488, found 385.1485.

17 20 3 4 3

Molecules 2017, 22, 1592

11 of 27

(

R)-2,4-Diamino-5-(3-triﬂuoromethylphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine

(

8f).

◦

−1

Method A. Light yellow solid 0.64 g, yield 68%; m.p. 126.0–126.8 C; IR: υmax/cm 3467 (NH), 3418

(

NH), 3336 (NH), 1650 (C=N), 1625 (C=N), 1593 (C=C), 1556 (C=C), 1523 (C=C), 1163 (C-O-C), 1118

1

C-O-C), 1106 (C-O-C), 1068 (C-O-C); H-NMR (CDCl )

δ

7.61 (s, 1H, Ar-H), 7.56–7.49 (m, 3H, Ar-H),

3

4

.73 (s, 2H, NH ), 4.55 (s, 2H, NH ), 4.36 (dd, J = 10.6, J = 4.2, 1H, OCH ), 4.31–4.26 (m, 1H, CH), 4.22

2 2 1 2 2

(

dd, J = 10.6, J = 5.8, 1H, OCH ), 3.96 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.70 (dd, J = 8.4, J = 6.0, 1H,

1 2 2 1 2 2 1 2

+

OCH ), 1.32 (s, 3H, CH ), 1.25 (s, 3H, CH ); ES-MS 385.1 (M + H) ; HRMS Calcd. for C H F N O

3

2

3

17 20

3

4

3

85.1488, found 385.1491.

(

R)-2,4-Diamino-5-(4-triﬂuoromethoxyphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (8g).

◦

−1

Method A. Yellow solid 0.77 g, yield 70%; m.p. 37.1–38.0 C; IR: υmax/cm 3482 (NH), 3368 (NH),

1

611 (C=N), 1560 (C=C), 1510 (C=C), 1374 (C-F), 1161 (C-O-C), 1092 (C-O-C), 1052 (C-O-C); H-NMR

(

CDCl₃) δ 7.35 (d, J = 8.4, 2H, Ar-H), 7.24 (d, J = 8.4, 1H, Ar-H), 4.71 (s, 2H, NH ), 4.54 (s, 2H, NH ),

2 2

4

3

.38 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.32–4.27 (m, 1H, CH), 4.21 (dd, J = 10.8, J = 5.6, 1H, OCH ),

1 2 2 1 2 2

.96 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.72 (dd, J = 8.4, J = 6.4, 1H, OCH ), 1.32 (s, 3H, CH ), 1.23 (s,

1

2

1

2

3

+

H, CH ); ES-MS 401.1(M + H) ; HRMS Calcd. for C H F N O 401.1437, found 401.1434.

3

17 20

3

4

(

R)-2,4-Diamino-5-(3-triﬂuoromethoxyphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (8h).

◦

−1

Method A. Yellow solid 0.72 g, yield 66%; m.p. 36.2–37.1 C; IR: υmax/cm 3488 (NH), 3361 (NH),

1

611 (C=N), 1560 (C=C), 1379 (C-F), 1158 (C-O-C), 1082 (C-O-C), 1047 (C-O-C); H-NMR (CDCl )

δ

3

7.41 (t, J = 8.0, 1H, Ar-H), 7.28–7.26 (m, 1H Ar-H), 7.21 (s, 1H, Ar-H), 7.14 (d, J = 8.4, 1H, Ar-H), 4.73

(

s, 2H, NH ), 4.58 (s, 2H, NH ), 4.36 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.32–4.26 (m, 1H, CH), 4.21

2

1

2

dd, J₁= 10.8, J = 6.0, 1H, OCH ), 3.97 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.72 (dd, J = 8.4, J = 6.0, 1H,

2

1

2

1

2

+

OCH ), 1.32 (s, 3H, CH ), 1.27 (s, 3H, CH ); ES-MS 401.1 (M + H) ; HRMS Calcd. for C H F N O

4

2

3

17 20

3

4

01.1437, found 401.1440.

(

R)-2,4-Diamino-5-(4-cyanophenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (8i). Method A.

◦

−1

White solid 0.62 g, yield 67%; m.p. 209.3–209.9 C; IR: υmax/cm 3482 (NH), 3456 (NH), 3370 (NH),

3

332 (NH), 2228 (CN), 1655 (C=N), 1621 (C=N), 1585 (C=C), 1564 (C=C), 1157 (C-O-C), 1091 (C-O-C),

1

051 (C-O-C); H-NMR (CDCl )

δ

7.68 (d, J = 8.4, 2H, Ar-H), 7.47 (d, J = 8.4, 2H Ar-H), 4.76 (s, 2H, NH₂),

3

4

.57 (s, 2H, NH ), 4.36 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.32–4.27 (m, 1H, CH), 4.23 (dd, J₁= 10.8

,

2

1

2

J₂= 6.0, 1H, OCH ), 3.97 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.70 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.32

2

1

2

1

2

+

(

s, 3H, CH ), 1.28 (s, 3H, CH ); ES-MS 342.1 (M + H) ; HRMS Calcd. for C H N O 342.1566, found

3 3 17 20 5 3

3

42.1570.

(

R)-2,4-Diamino-5-(4-methoxyphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (8j). Method

◦

−1

A. White solid 0.64 g, yield 68%; m.p. 119.7–120.2 C; IR: υmax/cm 3456 (NH), 3348 (NH), 1643

C=N), 1617(C=N), 1594 (C=C), 1560 (C=C), 1512 (C=C), 1171 (C-O-C), 1082 (C-O-C), 1050 (C-O-C);

(

1

H-NMR (CDCl₃)

δ 7.21 (d, J = 8.8, 2H, Ar-H), 6.93 (d, J = 8.8, 2H, Ar-H), 4.65 (s, 2H, NH ), 4.53 (s, 2H,

2

NH ), 4.37 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.32–4.26 (m, 1H, CH), 4.19 (dd, J = 10.8, J = 6.0, 1H,

2

1

2

1

2

OCH ), 3.96 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.82 (s, 3H, OCH ), 3.74 (dd, J = 8.4, J = 6.4, 1H, OCH ),

2

1

2

3

1

2

+

1

.32 (s, 3H, CH ), 1.28 (s, 3H, CH ); ES-MS 347.2 (M + H) ; HRMS Calcd. for C H N O 347.1719,

3 3 17 23 4 4

found 347.1723.

(

R)-2,4-Diamino-5-(3,4,5-trimethoxyphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine

(

8k).

◦

−1

Method A. White solid 0.70 g, yield 63%; m.p. 183.8–184.1 C; IR: υmax/cm 3458 (NH), 3421 (NH),

3

339 (NH), 1661 (C=N), 1631(C=N), 1588 (C=C), 1557 (C=C), 1511 (C=C), 1164 (C-O-C), 1128 (C-O-C),

1

2

6

074 (C-O-C); H-NMR (CDCl )

δ

6.54 (s, 2H, Ar-H), 4.67 (s, 2H, NH ), 4.62 (s, 2H, NH ), 4.38–4.31 (m,

3

2

H, OCH ), 4.27–4.20 (m, 1H, CH), 4.01 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.87 (s, 3H, OCH ), 3.84 (s,

2

1

2

3

H, OCH₃

×

2), 3.77 (dd, J = 8.0, J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ), 1.28 (s, 3H, CH ); ES-MS 407.2

1 2 2 3 3

+

(M + H) ; HRMS Calcd. for C H N O 407.1931, found 407.1927.

19 27 4 6

(

R)-2,4-Diamino-5-[3-(2,2,2-triﬂoroethoxymethyl)phenyl]-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]-pyrimidine

◦ −1

8l). Method A. Yellow solid 0.72 g, yield 62%; m.p. 32.2–33.2 C; IR: υmax/cm 3485 (NH), 3367

Molecules 2017, 22, 1592

12 of 27

(

NH), 1609 (C=N), 1559 (C=C), 1491 (C=C), 1373 (C-F), 1159 (C-O-C), 1093 (C-O-C), 1049 (C-O-C);

1

H-NMR (CDCl ) δ 7.40 (t, J = 7.6, 1H, Ar-H), 7.30–7.27 (m, 3H, Ar-H), 4.68 (s, 4H, CH , NH ), 4.56 (s,

3

2

H, NH ), 4.35 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.32–4.26 (m, 1H, CH), 4.22 (dd, J = 10.4, J₂= 5.6,

2 1 2 2 1

1

H, OCH ), 3.96 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.87 (q, J = 8.8, 2H, CF CH ), 3.73 (dd, J = 8.0,

2

1

2

3

2

+

1

J = 5.8, 1H, OCH ), 1.31 (s, 3H, CH ), 1.25 (s, 3H, CH ); ES-MS 429.2(M + H) ; HRMS Calcd. for

2

3

+

C H F N O 429.1750, found 429.1747.

19

24

3

4

(

R)-2,4-Diamino-5-[3,5-dimethyl-4-(N-methoxyaminosulfonyl)phenyl]-6-[4-(2,2-dimethyl-1,3-dioxolane)-

◦

methoxy]pyrimidine (8m). Method A. Light yellow solid 0.69 g, yield 56%; m.p. 157.7–158.8 C; IR:

υmax/cm 3485 (NH), 3446 (NH), 3347 (NH), 3223 (NH), 1640 (C=N), 1617 (C=N), 1591 (C=C), 1562

(

−

1

C=C), 1490 (C=C), 1170 (C-O-C), 1096 (C-O-C), 1053 (C-O-C); H-NMR (CDCl )

δ

7.43 (s, 1H, NH),

3

7

.18 (s, 2H, Ar-H), 4.74 (s, 2H, NH ), 4.61 (s, 2H, NH ), 4.35 (dd, J = 10.2, J = 5.0, 1H, OCH ), 4.32–4.27

2 2 1 2 2

(

m, 1H, CH), 4.24 (dd, J = 10.2, J = 5.0, 1H, OCH ), 3.99 (dd, J = 8.2, J = 6.2, 1H, OCH ), 3.75–3.72

1 2 2 1 2 2

m, 4H, OCH and OCH ), 2.69 (s, 6H, CH

×

2), 1.33 (s, 3H, CH ), 1.29 (s, 3H, CH ); ES-MS 454.2

3 3

3

2

3

+

M + H) ; HRMS Calcd. for C H N O S 454.1760, found 454.1756.

19

28

5

6

(

R)-2,4-Diamino-5-[4-(morpholine-4-carbonyl)phenyl]-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine

8n). Method A. Yellow solid 0.90 g, yield 77%; m.p. 189.4–190.5 C; IR: υmax/cm 3492 (NH), 3430

◦

−1

(

NH), 3334 (NH), 1659 (C=N), 1611(C=N), 1588 (C=C), 1560 (C=C), 1516 (C=C), 1488 (C=C), 1156

1

(C-O-C), 1114 (C-O-C), 1070 (C-O-C), 1051(C-O-C); H-NMR (CDCl )

δ

7.44 (d, J = 8.4, 2H, Ar-H), 7.38

3

(

d, J = 8.4, 2H, Ar-H), 4.71 (s, 2H, NH ), 4.57 (s, 2H, NH ), 4.35 (dd, J = 10.8, J = 4.2, 1H, OCH ),

2 2 1 2 2

4

3

.32–4.29 (m, 1H, CH), 4.22 (dd, J = 10.8, J = 5.6, 1H, OCH ), 3.97 (dd, J = 8.4, J = 6.4, 1H, OCH ),

1 2 2 1 2 2

.82–3.51 (m, 8H, CH₂

×

4), 3.73 (dd, J = 8.0, J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ), 1.27 (s, 3H, CH );

1 2 2 3 3

+

ES-MS 430.2 (M + H) ; HRMS Calcd. for C H N O 430.2090, found 430.2092.

21

28

5

(

R)-2,4-Diamino-5-[4-(3-triﬂoromethoxyanilinocarbonyl)phenyl]-6-[4-(2,2-dimethyl-1,3-dioxolane)-methoxy]

◦

−1

pyrimidine (8o). Method A. White solid 0.73 g, yield 52%; m.p. 122.9–124.5 C; IR: υmax/cm 3496

(

NH), 3340 (NH), 3192 (CONH), 1655 (C=N), 1607 (C=C), 1565 (C=C), 1492 (C=C), 1156 (C-O-C), 1082

1

C-O-C); H-NMR (CDCl )

δ

8.07 (s, 1H, NH), 7.85 (d, J = 8.4, 2H, Ar-H), 7.71 (s, 1H, Ar-H), 7.54 (dd,

3

J₁= 8.0, J = 1.2, 1H, Ar-H), 7.45 (d, J = 8.4, 2H, Ar-H), 7.38 (t, J = 8.0, 1H, Ar-H), 7.02 (d, J = 8.0, 1H,

2

Ar-H), 4.74 (s, 2H, NH ), 4.58 (s, 2H, NH ), 4.35 (dd, J = 9.6, J = 3.8, 1H, OCH ), 4.32–4.29 (m, 1H,

2

1

2

CH), 4.27 (dd, J = 9.6, J = 4.6, 1H, OCH ), 3.99 (dd, J = 8.4, J = 6.0, 1H, OCH ), 3.73 (dd, J = 8.2,

1

2

1

2

1

+

J = 5.4, 1H, OCH ), 1.30 (s, 3H, CH ), 1.29 (s, 3H, CH ); ES-MS 520.2 (M + H) ; HRMS Calcd. for

2

3

+

C H F N O 520.1808, found 520.1810.

2

4

25

3

5

(

R)-2,4-Diamino-5-(4-methoxycarbonylphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (8p).

◦

−1

Method B. White solid 0.64 g, yield 63%; m.p. 151.3–152.4 C; IR: υmax/cm 3500 (NH), 3443

(

NH), 3391 (NH), 3335 (NH), 1709 (C=O), 1657 (C=N), 1608 (C=N), 1568 (C=C), 1556 (C=C), 1513

1

C=C), 1194 (C-O-C), 1180 (C-O-C), 1152 (C-O-C), 1116 (C-O-C), 1067 (C-O-C); H-NMR (CDCl )

δ 8.05

3

dd, J = 6.8, J = 1.6, 2H, Ar-H), 7.42 (dd, J = 6.8, J = 1.6, 2H, Ar-H), 4.72 (s, 2H, NH ), 4.58 (s, 2H,

1

2

1

2

NH ), 4.36 (dd, J = 10.4, J = 4.0, 1H, OCH ), 4.31–4.26 (m, 1H, CH), 4.22 (dd, J = 10.4, J = 6.0, 1H,

2

1

2

1

2

OCH ), 3.96 (dd, J = 8.0, J = 6.0, 1H, OCH ), 3.93 (s, 3H, OCH ), 3.72 (dd, J = 8.4, J = 6.0, 1H, OCH ),

2

1

2

3

1

2

+

1

.32 (s, 3H, CH ), 1.27 (s, 3H, CH ); ES-MS 375.2(M + H) ; HRMS Calcd. for C H N O 375.1668,

3 3 18 23 4 5

found 375.1664.

(

R)-2,4-Diamino-5-(3-methoxycarbonylphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (8q).

◦

−1

Method B. White solid 0.66 g, yield 65%; m.p. 181.4–181.6 C; IR: υmax/cm 3477 (NH), 3373

(

NH), 3326 (NH), 1708 (C=O), 1651 (C=N), 1626 (C=N), 1597 (C=C), 1558 (C=C), 1490 (C=C), 1158

1

C-O-C), 1115 (C-O-C), 1084 (C-O-C), 1049 (C-O-C); H-NMR (CDCl )

δ 8.00 (t, J = 1.6, 1H, Ar-H), 7.96

3

dt, J = 7.6, J = 1.6, 1H, Ar-H), 7.52 (dt, J = 7.6, J = 1.6, 1H, Ar-H), 7.47 (t, J = 7.6, 1H, Ar-H), 4.71

1

2

1

2

s, 2H, NH ), 4.54 (s, 2H, NH ), 4.37 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.30–4.25 (m, 1H, CH), 4.19

2

1

2

dd, J₁= 10.8, J = 6.0, 1H, OCH ), 3.96 (dd, J = 8.4, J = 6.0, 1H, OCH ), 3.92 (s, 3H, OCH ), 3.73 (dd,

2

1

2

3

Molecules 2017, 22, 1592

13 of 27

+

J₁= 8.4, J = 6.0, 1H, OCH ), 1.31 (s, 3H, CH ), 1.25 (s, 3H, CH ); ES-MS 375.2(M + H) ; HRMS Calcd.

2

3

+

for C H N O 375.1668, found 375.1670.

18

23

4

5

(

S)-2,4-Diamino-5-(4-chlorophenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (9a). Method A.

◦

−1

White solid 0.58 g, yield 61%; m.p. 171.9–172.1 C; IR: υmax/cm 3475 (NH), 3454 (NH), 3396 (NH),

3

315 (NH), 1649 (C=N), 1620 (C=N), 1593 (C=C), 1549 (C=C), 1479 (C=C), 1160 (C-O-C), 1141 (C-O-C),

1

089(C-O-C), 794 (C-Cl); H-NMR (CDCl )

δ

7.36 (d, J = 8.2, 2H, Ar-H), 7.25 (d, J = 8.2, 2H, Ar-H), 4.69

3

(

s, 2H, NH ), 4.53 (s, 2H, NH ), 4.36 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.31–4.26 (m, 1H, CH), 4.20 (dd,

2 2 1 2 2

J = 10.8, J = 5.6, 1H, OCH ), 3.97 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.72 (dd, J = 8.4, J = 6.0, 1H,

1

2

1

2

1

2

+

OCH ), 1.32 (s, 3H, CH ), 1.28 (s, 3H, CH ); ES-MS 351.1 (M + H) ; HRMS Calcd. for C H ClN O

3

2

3

16 20

4

3

51.1224, found 351.1220.

(

S)-2,4-Diamino-5-(4-ﬂuorophenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (9b). Method A.

◦

−1

White solid 0.57 g, yield 62%; m.p. 164.1–164.5 C; IR: υmax/cm 3527 (NH), 3482 (NH), 3370 (NH),

3

312 (NH), 1646 (C=N), 1626 (C=N), 1593 (C=C), 1558 (C=C), 1512 (C=C), 1484 (C=C), 1380 (C-F), 1159

1

(

C-O-C), 1130 (C-O-C), 1091 (C-O-C); H-NMR (CDCl )

δ

7.26 (dd, J = 8.8, J = 5.2, 2H, Ar-H), 7.08 (t,

3

1

2

J = 8.8, 2H, Ar-H), 4.67 (s, 2H, NH ), 4.50 (s, 2H, NH ), 4.36 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.31–4.26

2

1

2

(

m, 1H, CH), 4.20 (dd, J = 10.8, J = 5.6, 1H, OCH ), 3.96 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.72 (dd,

1 2 2 1 2 2

+

J₁= 8.4, J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ), 1.27 (s, 3H, CH ); ES-MS 335.2 (M + H) ; HRMS Calcd.

for C H FN O 335.1519, found 335.1521.

2

3

+

16

20

4

3

(

S)-2,4-Diamino-5-(3,4-dichlorophenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (9c). Method A.

◦

−1

White solid 0.58 g, yield 55%; m.p. 167.3–167.7 C; IR: υmax/cm 3487 (NH), 3434(NH), 3378 (NH),

3

318 (NH), 1650 (C=N), 1624 (C=N), 1600 (C=C), 1558 (C=C), 1487 (C=C), 1155 (C-O-C), 1093 (C-O-C),

1

1047 (C-O-C), 795 (C-Cl); H-NMR (CDCl )

δ

7.47 (d, J = 8.4, 1H, Ar-H), 7.44 (d, J = 2.0, 1H, Ar-H),

4.35 (dd, J = 10.4, J = 4.4,

3

7

.18 (dd, J = 8.4, J = 2.0, 1H, Ar-H), 4.71 (s, 2H, NH ), 4.54 (s, 2H, NH )

,

1

2

1

2

1

H, OCH ), 4.33–4.27 (m, 1H, CH), 4.22 (dd, J = 10.4, J = 5.2, 1H, OCH ), 3.99 (dd, J = 8.4, J = 6.0,

2 1 2 2 1 2

H, OCH ), 3.73 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.34 (s, 3H, CH ), 1.30 (s, 3H, CH ); ES-MS 385.1

2

1

2

3

+

(M + H) ; HRMS Calcd. for C H Cl N O 385.0834, found 385.0838.

16 19 2 4 3

(

S)-2,4-Diamino-5-(3-chloro-4-ﬂuorophenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine

(

9d).

◦

−1

Method A. White solid 0.56 g, yield 56%; m.p. 152.4–152.6 C; IR: υmax/cm 3527 (NH), 3477 (NH),

3

377 (NH), 3355 (NH), 1626 (C=N), 1598 (C=C), 1559 (C=C), 1482 (C=C), 1376 (C-F), 1163 (C-O-C),

1

128 (C-O-C), 1093 (C-O-C), 1043 (C-O-C), 798 (C-Cl); H-NMR (CDCl )

H, Ar-H), 7.21–7.14 (m, 2H, Ar-H), 4.71 (s, 2H, NH ), 4.53 (s, 2H, NH ), 4.35 (dd, J = 10.6, J = 4.2,

H, OCH ), 4.32–4.27 (m, 1H, CH), 4.22 (dd, J = 10.6, J = 5.4, 1H, OCH ), 3.98 (dd, J = 8.4, J = 6.4,

H, OCH ), 3.72 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.33 (s, 3H, CH ), 1.29 (s, 3H, CH ); ES-MS 369.1

δ

7.37 (dd, J = 7.0, J = 1.8,

3

1

2

1

2

1

2

1

2

1

2

3

+

(M + H) ; HRMS Calcd. for C H ClFN O , 369.1130, found 369.1132.

16 19 4 3

(

S)-2,4-Diamino-5-(4-triﬂuoromethylphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine

(

9e).

◦

−1

Method A. Light yellow solid 0.64 g, yield 61%; m.p. 130.0–131.6 C; IR: υmax/cm 3467 (NH), 3418

(

NH), 3336 (NH), 1650 (C=N), 1625 (C=N), 1593 (C=C), 1556 (C=C), 1523 (C=C), 1163 (C-O-C), 1118

1

C-O-C), 1106 (C-O-C), 1068 (C-O-C); H-NMR (CDCl )

δ 7.64 (d, J = 8.0, 2H, Ar-H), 7.46 (d, J = 8.0, 2H,

3

Ar-H), 4.72 (s, 2H, NH ), 4.55 (s, 2H, NH ), 4.37 (dd, J = 10.8, J = 4.2, 1H, OCH ), 4.32–4.27 (m, 1H,

2

1

2

CH), 4.22 (dd, J = 10.8, J = 5.6, 1H, OCH ), 3.97 (dd, J = 8.4, J = 6.0, 1H, OCH ), 3.71 (dd, J = 8.4,

1

2

1

2

1

+

J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ), 1.24 (s, 3H, CH ); ES-MS 385.1 (M + H) ; HRMS Calcd. for

2

3

+

C H F N O 385.1488, found 385.1490.

1

7

20

3

4

3

(

S)-2,4-Diamino-5-(3-triﬂuoromethylphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine

(

9f).

◦

−1

Method A. Light yellow solid 0.74 g, yield 71%; m.p. 126.3–126.5 C; IR: υmax/cm 3512 (NH), 3420

(

NH), 3382 (NH), 3327 (NH), 1650 (C=N), 1612 (C=N), 1593 (C=C), 1559 (C=C), 1171 (C-O-C), 1128

1

C-O-C), 1094 (C-O-C), 1072 (C-O-C), 1046 (C-O-C); H-NMR (CDCl )

δ

7.61 (s, 1H, Ar-H), 7.56–7.49

3

m, 3H, Ar-H), 4.73 (s, 2H, NH ), 4.55 (s, 2H, NH ), 4.36 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.31–4.25

2

1

2

m, 1H, CH), 4.21 (dd, J = 10.8, J = 5.6, 1H, OCH ), 3.97 (dd, J = 8.2, J = 6.0, 1H, OCH ), 3.71 (dd,

1

2

1

2

Molecules 2017, 22, 1592

14 of 27

+

J₁= 8.2, J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ), 1.25 (s, 3H, CH ); ES-MS 385.1 (M + H) ; HRMS Calcd.

2

3

+

for C H F N O 385.1488, found 385.1491.

17

20

3

4

3

(

S)-2,4-Diamino-5-(4-triﬂuoromethoxyphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (9g).

◦

−1

Method A. Yellow solid 0.80 g, yield 73%; m.p. 36.7–37.7 C; IR: υmax/cm 3487 (NH), 3358 (NH),

1

612 (C=N), 1559 (C=C), 1510 (C=C), 1374 (C-F), 1160 (C-O-C), 1092 (C-O-C), 1053 (C-O-C); H-NMR

(

CDCl₃) δ 7.35 (d, J = 8.4, 2H, Ar-H), 7.24 (d, J = 8.4, 1H, Ar-H), 4.70 (s, 2H, NH ), 4.53 (s, 2H, NH ),

2 2

4

3

.38 (dd, J = 11.2, J = 4.0, 1H, OCH ), 4.32–4.26 (m, 1H, CH), 4.21 (dd, J = 11.2, J = 5.4, 1H, OCH ),

1 2 2 1 2 2

.96 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.72 (dd, J = 8.2, J = 6.2, 1H, OCH ), 1.32 (s, 3H, CH ), 1.24 (s,

1

2

1

2

3

+

H, CH ); ES-MS 401.1(M + H) ; HRMS Calcd. for C H F N O 401.1437, found 401.1440.

3

17 20

3

4

(

S)-2,4-Diamino-5-(3-triﬂuoromethoxyphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (9h).

◦

−1

Method A. Yellow solid 0.73 g, yield 67%; m.p. 33.7–34.7 C; IR: υmax/cm 3490 (NH), 3360 (NH),

1

611 (C=N), 1561 (C=C), 1379 (C-F), 1158 (C-O-C), 1082 (C-O-C), 1047 (C-O-C); H-NMR (CDCl )

δ

7.41

3

(

t, J = 8.0, 1H, Ar-H), 7.28–7.26 (m, 1H Ar-H), 7.21 (s, 1H, Ar-H) 7.14 (dt, J = 8.4, J = 1.2, 1H, Ar-H),

1 2

4

.75 (s, 2H, NH ), 4.60 (s, 2H, NH ), 4.36 (dd, J = 10.8, J = 4.2, 1H, OCH ), 4.32–4.26 (m, 1H, CH), 4.21

2 2 1 2 2

(

dd, J = 10.8, J = 5.6, 1H, OCH ), 3.97 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.72 (dd, J = 8.4, J = 6.0, 1H,

1 2 2 1 2 2 1 2

+

OCH ), 1.32 (s, 3H, CH ), 1.27 (s, 3H, CH ); ES-MS 401.1(M + H) ; HRMS Calcd. for C H F N O

4

2

3

17 20

3

4

01.1437, found 401.1435.

(

S)-2,4-Diamino-5-(4-cyanophenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (9i). Method A.

◦

−1

White solid 0.66 g, yield 71%; m.p. 221.4–221.9 C; IR: υmax/cm 3482 (NH), 3456 (NH), 3371 (NH),

3

332 (NH), 2228 (CN), 1654 (C=N), 1620 (C=N), 1586 (C=C), 1564 (C=C), 1157 (C-O-C), 1091 (C-O-C),

1

051 (C-O-C); H-NMR (CDCl )

δ

7.67 (d, J = 8.4, 2H, Ar-H), 7.47 (d, J = 8.4, 2H Ar-H), 4.75 (s, 2H, NH₂),

3

4

.57 (s, 2H, NH ), 4.36 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.32–4.27 (m, 1H, CH), 4.23 (dd, J₁= 10.4

,

2

1

2

J₂= 4.2, 1H, OCH ), 3.97 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.70 (dd, J = 8.4, J = 6.0, 1H, OCH ),

2

1

2

1

2

+

1

.32 (s, 3H, CH ), 1.28 (s, 3H, CH ); ES-MS 342.1 (M + H) ; HRMS Calcd. for C H N O 342.1566,

3 3 17 20 5 3

found 342.1564.

(

S)-2,4-Diamino-5-(4-methoxyphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (9j). Method A.

◦

−1

White solid 0.57 g, yield 60%; m.p. 120.1–121.3 C; IR: υmax/cm 3458 (NH), 3349 (NH), 1620 (C=N),

1

594 (C=C), 1558 (C=C), 1513 (C=C), 1157 (C-O-C), 1085 (C-O-C), 1046 (C-O-C); H-NMR (CDCl )

δ

3

7

.21 (d, J = 8.8, 2H, Ar-H), 6.93 (d, J = 8.8, 2H, Ar-H), 4.65 (s, 2H, NH ), 4.53 (s, 2H, NH ), 4.37 (dd,

2 2

J₁= 10.8, J = 4.0, 1H, OCH ), 4.32–4.26 (m, 1H, CH), 4.19 (dd, J = 10.8, J = 6.0, 1H, OCH ), 3.96 (dd,

2

1

2

J₁= 8.4, J = 6.0, 1H, OCH ), 3.82 (s, 3H, OCH ), 3.74 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ),

2

3

1

2

3

+

1

.28 (s, 3H, CH ); ES-MS 347.2 (M + H) ; HRMS Calcd. for C H N O 347.1719, found 347.1721.

3 17 23 4 4

(

S)-2,4-Diamino-5-(3,4,5-trimethoxyphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine

(

9k).

◦

−1

Method A. White solid 0.61 g, yield 55%; m.p. 181.7–183.0 C; IR: υmax/cm 3458 (NH), 3421 (NH),

3

339 (NH), 1660 (C=N), 1631 (C=N), 1586 (C=C), 1557 (C=C), 1510 (C=C), 1163 (C-O-C), 1129 (C-O-),

1

1075 (C-O-C); H-NMR (CDCl )

δ

6.55 (s, 2H, Ar-H), 4.66 (s, 2H, NH ), 4.61 (s, 2H, NH ), 4.38–4.31 (m,

3

2

H, OCH ), 4.2–4.21 (m, 1H, CH), 4.00 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.87 (s, 3H, OCH ), 3.84 (s, 6H,

2 1 2 2 3

OCH₃

×

2), 3.77 (dd, J = 8.0, J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ), 1.28 (s, 3H, CH ); ES-MS 407.2

1 2 2 3 3

+

(M + H) ; HRMS Calcd. for C H N O 407.1931, found 407.1933.

19 27 4 6

(

S)-2,4-Diamino-5-[3-(2,2,2-triﬂuoroethoxymethyl)phenyl]-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]-

◦

−1

pyrimidine (9l). Method A. Yellow solid 0.73 g, yield 62%; m.p. 32.8–33.7 C; IR: υmax/cm 3482

(

NH), 3372 (NH), 1611 (C=N), 1559 (C=C), 1377 (C-F), 1159 (C-O-C), 1121 (C-O-C), 1093 (C-O-C), 1049

1

C-O-C); H-NMR (CDCl )

δ 7.40 (t, J = 7.6, 1H, Ar-H), 7.30–7.27 (m, 3H, Ar-H), 4.68 (s, 4H, OCH2

3

and NH ), 4.55 (s, 2H, NH ), 4.35 (dd, J = 10.4, J = 4.0, 1H, OCH ), 4.31–4.26 (m, 1H, CH), 4.22 (dd,

2

1

2

J₁= 10.8

, J₂= 5.6, 1H, OCH ), 3.96 (dd, J = 8.4, J = 6.0, 1H, OCH ), 3.86 (q, J = 8.8, 2H, CF CH ), 3.73

2 1 2 2 3 2

+

(dd, J₁= 8.4, J = 6.0, 1H, OCH ), 1.31 (s, 3H, CH ), 1.25 (s, 3H, CH ); ES-MS 429.2(M + H) ; HRMS

2

3

+

Calcd. for C H F N O 429.1750, found 429.1752.

19

24

3

4

Molecules 2017, 22, 1592

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(

S)-2,4-Diamino-5-[3,5-dimethyl-4-(N-methoxyaminosulfonyl)phenyl]-6-[4-(2,2-dimethyl-1,3-dioxolane)-

◦

methoxy]pyrimidine (9m). Method A. Light yellow solid 0.75 g, yield 61%; m.p. 160.5–160.8 C; IR:

υmax/cm 3484 (NH), 3446 (NH), 3347 (NH), 3223 (SO NH), 1641 (C=N), 1619 (C=N), 1591 (C=C),

1

−

1

2

1

562 (C=C), 1492 (C=C), 1173 (C-O-C), 1149 (C-O-C), 1099 (C-O-C), 1054 (C-O-C); H-NMR (CDCl )

δ

3

7

.42 (s, 1H, NH), 7.18 (s, 2H, Ar-H), 4.73 (s, 2H, NH ), 4.60 (s, 2H, NH ), 4.35 (dd, J = 10.4, J = 4.0, 1H,

2 2 1 2

OCH ), 4.32–4.27 (m, 1H, CH), 4.24 (dd, J = 10.4, J = 4.8, 1H, OCH ), 3.99 (dd, J = 8.2, J = 6.2, 1H,

2

1

2

1

2

OCH ), 3.75–3.72 (m, 4H, OCH and OCH ), 2.69 (s, 6H, CH

×

2), 1.33 (s, 3H, CH ), 1.29 (s, 3H, CH );

3 3

2

3

2

3

+

ES-MS 454.2 (M + H) ; HRMS Calcd. for C H N O S 454.1760, found 454.1762.

19

28

5

6

(

S)-2,4-Diamino-5-[4-(morpholino-4-carbonyl)phenyl]-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine

9n). Method A. White solid 0.64 g, yield 55%; m.p. 186.1–187.2 C; IR: υmax/cm 3492 (NH), 3430

◦

−1

NH), 3332 (NH), 1659 (C=N), 1610 (C=N), 1588 (C=C), 1558 (C=C), 1516 (C=C), 1488 (C=C), 1156

1

C-O-C), 1114 (C-O-C), 1070 (C-O-C), 1050 (C-O-C); H-NMR (CDCl )

δ 7.45 (d, J = 8.4, 2H, Ar-H),

3

7

4

3

.38 (d, J = 8.4, 2H, Ar-H), 4.82 (s, 2H, NH ), 4.67 (s, 2H, NH ), 4.38 (dd, J = 10.8, J = 4.4, 1H, OCH ),

2 2 1 2 2

.32–4.28 (m, 1H, CH), 4.23 (dd, J = 10.8, J = 5.6, 1H, OCH ), 3.97 (dd, J = 8.2, J = 6.2, 1H, OCH ),

1

2

1

2

.82–3.51 (m, 8H, CH₂

×

4), 3.72 (dd, J = 8.4, J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ), 1.28 (s, 3H, CH );

1 2 2 3 3

+

ES-MS 430.2 (M + H) ; HRMS Calcd. for C H N O 430.2090, found 430.2088.

21

28

5

(

S)-2,4-Diamino-5-[4-(3-triﬂuoromethoxyanilinocarbonyl)phenyl]-6-[4-(2,2-dimethyl-1,3-dioxolane)-methoxy]

◦

−1

pyrimidine (9o). Method A. White solid 0.74 g, yield 52%; m.p. 83.7–85 C; IR: υmax/cm 3488 (NH),

3

351 (NH), 3202 (CONH), 1657 (C=N), 1606 (C=C), 1565 (C=C), 1548 (C=C), 1492 (C=C), 1156 (C-O-C),

1

1082 (C-O-C); H-NMR (CDCl )

δ

8.04 (s, 1H, NH), 7.85 (d, J = 8.4, 2H, Ar-H), 7.71 (s, 1H, Ar-H),

3

7

.54 (dd, J = 8.0, J = 1.2, 1H, Ar-H), 7.45 (d, J = 8.4, 2H, Ar-H), 7.39 (t, J = 8.0, 1H, Ar-H), 7.02 (dt,

1 2

J₁= 8.0, J₂= 1.0, 1H, Ar-H), 4.74 (s, 2H, NH ), 4.59 (s, 2H, NH ), 4.35 (dd, J = 10.0, J = 4.0, 1H, OCH ),

2 2 1 2 2

4

3

.32–4.29 (m, 1H, CH), 4.26 (dd, J = 10.0, J = 4.8, 1H, OCH ), 3.99 (dd, J = 8.4, J = 6.0, 1H, OCH ),

1 2 2 1 2 2

+

.73 (dd, J = 8.2, J = 5.4, 1H, OCH ), 1.30 (s, 3H, CH ), 1.29 (s, 3H, CH ); ES-MS 520.2 (M + H) ;

1

2

3

+

HRMS Calcd. for C H F N O 520.1808, found 520.1811.

2

4

25

3

5

(

S)-2,4-Diamino-5-(4-methoxycarbonylphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (9p).

◦

−1

Method B. White solid 0.70 g, yield 69%; m.p. 146.3–147.4 C; IR: υmax/cm 3501 (NH), 3442

(

NH), 3392 (NH), 3336 (NH), 1708 (C=O), 1658 (C=N), 1610 (C=N), 1555 (C=C), 1515 (C=C), 1153

1

C-O-C), 1115 (C-O-C), 1067 (C-O-C); H-NMR (CDCl )

δ 8.05 (d, J = 8.4, 2H, Ar-H), 7.29 (d, J = 8.4, 2H,

3

Ar-H), 4.72 (s, 2H, NH ), 4.58 (s, 2H, NH ), 4.36 (dd, J = 10.6, J = 4.2, 1H, OCH ), 4.31–4.26 (m, 1H,

2

1

2

CH), 4.22 (dd, J = 10.6, J = 5.8, 1H, OCH ), 3.96 (dd, J = 8.4, J = 6.0, 1H, OCH ), 3.93 (s, 3H, OCH ),

1

2

1

2

3

+

3

.71 (dd, J = 8.0, J = 6.0, 1H, OCH ), 1.32 (s, 3H, CH ), 1.27 (s, 3H, CH ); ES-MS 375.2(M + H) ; HRMS

1 2 2 3 3

+

Calcd. for C H N O 375.1668, found 375.1670.

18

23

4

5

(

S)-2,4-Diamino-5-(3-methoxycarbonylphenyl)-6-[4-(2,2-dimethyl-1,3-dioxolane)methoxy]pyrimidine (9q).

◦

−1

Method B. White solid 0.80 g, yield 78%; m.p. 179.2–180.3 C; IR: υmax/cm 3480 (NH), 3371

(

NH), 3326 (NH), 1708 (C=O), 1652 (C=N), 1626 (C=N), 1601 (C=C), 1592 (C=C), 1557 (C=C), 1158

1

C-O-C), 1115 (C-O-C), 1084 (C-O-C), 1049 (C-O-C); H-NMR (CDCl )

δ 8.00 (t, J = 1.6, 1H, Ar-H), 7.96

3

dt, J = 7.6, J = 1.6, 1H, Ar-H), 7.52 (dt, J = 7.6, J = 1.6, 1H, Ar-H), 7.47 (t, J = 7.6, 1H, Ar-H), 4.71

1

2

1

2

s, 2H, NH ), 4.54 (s, 2H, NH ), 4.37 (dd, J = 10.8, J = 4.0, 1H, OCH ), 4.30–4.25 (m, 1H, CH), 4.19

2

1

2

dd, J₁= 10.8, J = 6.0, 1H, OCH ), 3.96 (dd, J = 8.4, J = 6.4, 1H, OCH ), 3.92 (s, 3H, OCH ), 3.72 (dd,

2

1

2

3

+

J₁= 8.4, J = 6.0, 1H, OCH ), 1.31 (s, 3H, CH ), 1.25 (s, 3H, CH ); ES-MS 375.2(M + H) ; HRMS Calcd.

for C H N O 375.1668, found 375.1666.

2

3

+

18

23

4

5

General Procedure for the Synthesis of Compounds 10a-q and 11a-q

The compounds 8a-q or 9a-q (1.42 mmol) was added to 0.25 M H SO (20 mL) and stirred at room

2

4

temperature overnight. After the completion of the reaction as indicated by TLC analysis, the reaction

solution was adjusted the pH to 9 and extracted with EtOAc (30 mL 3). The combined organic layers

were washed by H O and dried with Na SO , ﬁltered and concentrated. The residue was puriﬁed by

×

2

4

Molecules 2017, 22, 1592

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column chromatography on silica gel using CH Cl /CH OH (80:1, v/v) as the eluting solvent to the

2

3

desired compounds.

(

R)-2,4-Diamino-5-(4-chlorophenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10a). White solid 0.37 g, yield 84%;

◦

−1

m.p. 164.4–166.3 C; IR: υmax/cm 3443 (OH), 3396 (OH), 3335 (NH), 3224 (NH), 1658 (C=N), 1627

(

1

C=N), 1604 (C=C), 1591 (C=C), 1569 (C=C), 1555 (C=C), 1503 (C=C), 1483 (C=C), 796 (C-Cl); H-NMR

(

DMSO-d₆)

δ 7.38 (d, J = 8.4, 2H, Ar-H), 7.27 (d, J = 8.4, 2H, Ar-H), 6.02 (s, 2H, NH ), 5.63 (s, 2H, NH ),

2 2

4

.72 (d, J = 4.8, 1H, CHOH), 4.50 (t, J = 5.8, 1H, CH OH), 4.08 (d, J = 5.2, 2H, OCH ), 3.62–3.56 (m, 1H,

2

+

CH), 3.29–3.23 (m, 2H, OCH ); ES-MS 311.1 (M + H) ; HRMS Calcd. for C H ClN O 311.0911,

2

13 16

4

3

found 311.0909.

(

R)-2,4-Diamino-5-(4-ﬂuorophenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10b). White solid 0.30 g, yield 68%;

◦

−1

m.p. 122.4–122.8 C; IR: υmax/cm 3520 (OH), 3472 (OH), 3407 (NH), 3349 (NH), 1651 (C=N), 1617

(

1

C=N), 1592 (C=C), 1570 (C=C), 1484 (C=C), 1397 (C-F); H-NMR (DMSO-d )

δ

7.28 (dd, J = 8.8,

6

1

J₂= 5.6, 2H, Ar-H), 7.15 (t, J = 8.8, 2H, Ar-H), 5.99 (s, 2H, NH ), 5.57 (s, 2H, NH ), 4.72 (d, J = 4.8, 1H,

2

CHOH), 4.49 (t, J = 5.6, 1H, CH OH), 4.08 (d, J = 5.6, 2H, OCH ), 3.63–3.55 (m, 1H, CH), 3.31–3.24 (m,

2

+

2

H, OCH ); ES-MS 295.1 (M + H) ; HRMS Calcd. for C H FN O 295.1206, found 295.1204.

2 13 16 4 3

(

7

1

R)-2,4-Diamino-5-(3,4-dichlorophenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10c). White solid 0.32 g, yield

◦

−1

1%; m.p. 159.5–160.2 C; IR: υmax/cm 3504 (OH), 3476 (OH), 3344 (NH), 3229 (NH), 1650 (C=N),

1

619(C=N), 1593 (C=C), 1570 (C=C), 1490 (C=C), 792 (C-Cl); H-NMR (DMSO-d )

δ

7.56 (d, J = 8.0, 1H,

6

Ar-H), 7.47 (d, J = 2.0, 1H, Ar-H), 7.24 (dd, J =8.0, J = 2.0, 1H, Ar-H), 6.07 (s, 2H, NH ), 5.79 (s, 2H,

1

2

NH ), 4.74 (d, 1H, J = 4.8, CHOH), 4.52 (t, J = 5.8, 1H, CH OH), 4.14–4.06 (m, 2H, OCH ), 3.63–3.57 (m,

2

+

1

H, CH), 3.28–3.31 (m, 2H, OCH ); ES-MS 345.1 (M + H) ; HRMS Calcd. for C H Cl N O 345.0521,

2 13 15 2 4 3

found 345.0519.

(

R)-2,4-Diamino-5-(3-chloro-4-ﬂuorophenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10d). White solid 0.39 g,

◦

−1

yield 88%; m.p. 158.0–159.9 C; IR: υmax/cm 3484 (OH), 3437 (OH), 3318 (NH), 3209 (NH), 1627

(

1

C=N), 1586 (C=C), 1560 (C=C), 1349 (C-F), 801 (C-Cl); H-NMR (DMSO-d )

δ

7.40 (dd, J = 7.6, J₂= 2.4

,

6

1

H, Ar-H), 7.36 (t, J = 9.0, 1H, Ar-H), 7.23 (ddd, J = 8.4, J = 4.8, J = 2.0, 1H, Ar-H), 6.04 (s, 2H,

1 2 3

NH ), 5.74 (s, 2H, NH ), 4.74 (d, J = 4.8, 1H, CHOH), 4.51 (t, J = 5.8, 1H, CH OH), 4.13–4.05 (m, 2H,

2

+

OCH ), 3.63–3.56 (m, 1H, CH), 3.30–3.26 (m, 2H, OCH ); ES-MS 329.1(M + H) ; HRMS Calcd. for

C H ClFN O 329.0817, found 329.0820.

2

+

13

15

4

3

(

R)-2,4-Diamino-5-(4-triﬂuoromethylphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10e). White solid 0.37 g,

◦

−1

yield 83%; m.p. 170.5–171.0 C; IR: υmax/cm 3587 (OH), 3506 (OH), 3378 (NH), 3226 (NH), 1631

(

1

C=N), 1591 (C=C), 1559 (C=C), 1520 (C=C), 1322 (C-F); H-NMR (DMSO-d )

δ

7.67 (d, J = 8.4, 2H,

6

Ar-H), 7.50 (d, J = 8.0, 2H, Ar-H), 6.09 (s, 2H, NH ), 5.75 (s, 2H, NH ), 4.74 (d, J = 5.2, 1H, CHOH), 4.52

2

(

t, J = 5.8, 1H, CH OH), 4.15–4.07 (m, 2H, OCH ), 3.64–3.57 (m, 1H, CH), 3.24–3.30 (m, 2H, OCH );

2

+

ES-MS 345.1 (M + H) ; HRMS Calcd. for C H F N O 345.1175, found 345.1173.

14

16

3

4

3

(

R)-2,4-Diamino-5-(3-triﬂuoromethylphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10f). White solid 0.41 g,

◦

−1

yield 92%; m.p. 137.8–138.9 C; IR: υmax/cm 3521 (OH), 3474 (OH), 3349 (NH), 3235 (NH), 1650

(

1

C=N), 1621 (C=N), 1594 (C=C), 1569 (C=C), 1495 (C=C), 1341 (C-F); H-NMR (DMSO-d )

δ

7.58–7.55

6

(

m, 4H, Ar-H), 6.08 (s, 2H, NH ), 5.74 (s, 2H, NH ), 4.72 (d, 1H, J = 5.2, CHOH), 4.50 (t, J = 5.8, 1H,

2

CH OH), 4.11 (d, J = 4.8, 2H, OCH ), 3.62–3.56 (m, 1H, CH), 3.31–3.26 (m, 2H, OCH ); ES-MS 345.1

2

+

(

M + H) ; HRMS Calcd. for C H F N O 345.1175, found 345.1172.

14 16 3 4 3

(

R)-2,4-Diamino-5-(4-triﬂuoromethoxyphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10g). White solid 0.42 g,

◦

−1

yield 85%; m.p. 181.4–182.1 C; IR: υmax/cm 3579 (OH), 3457 (OH), 3341(NH), 3231 (NH), 1636

(

1

C=N), 1590 (C=C), 1557 (C=C), 1549 (C=C), 1501 (C=C), 1316 (C-F); H-NMR (DMSO-d )

δ

7.38 (d,

6

J = 8.4, 2H, Ar-H), 7.31 (d, J = 8.4, 2H, Ar-H), 6.06 (s, 2H, NH ), 5.69 (s, 2H, NH ), 4.77 (d, J = 5.2, 1H,

2

CHOH), 4.53 (t, J = 5.8, 1H, CH OH), 4.10 (d, J = 5.2, 2H, OCH ), 3.63–3.57 (m, 1H, CH), 3.32–3.23 (m,

2

+

2

H, OCH ); ES-MS 361.1 (M + H) ; HRMS Calcd. for C H F N O 361.1124, found 361.1120.

2 14 16 3 4 4

Molecules 2017, 22, 1592

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(

R)-2,4-Diamino-5-(3-triﬂuoromethoxyphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10h). White solid 0.44 g,

◦

−1

yield 89%; m.p. 58.5–59.6 C; IR: υmax/cm 3467 (OH), 3379 (OH), 3350 (NH), 1612 (C=N), 1562

(

1

C=C), 1495 (C=C), 1348 (C-F); H-NMR (DMSO-d )

δ

7.46 (t, J = 8.0, 2H, Ar-H), 7.31 (d, J = 8.0, 2H,

6

Ar-H), 7.23 (s, 1H, Ar-H), 7.20 (d, J = 8.0, 1H, Ar-H), 6.09 (s, 2H, NH ), 5.73 (s, 2H, NH ), 4.75 (d, J = 4.8,

2

1

H, CHOH), 4.52 (t, J = 5.6, 1H, CH OH), 4.10 (d, J = 5.2, 2H, OCH ), 3.63–3.56 (m, 1H, CH), 3.31–3.24

2

+

(m, 2H, OCH ); ES-MS 361.1 (M + H) ; HRMS Calcd. for C H F N O 361.1124, found 361.1128.

2 14 16 3 4 4

(

R)-2,4-Diamino-5-(4-cyanophenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10i). White solid 0.39 g, yield 88%;

◦

−1

m.p. 199.7–200.8 C; IR: υmax/cm 3459 (OH), 3382 (NH), 2228(CN), 1658 (C=N), 1624(C=N), 1592

(

1

C=C), 1563 (C=C), 1549 (C=C), 1510 (C=C); H-NMR (DMSO-d )

δ

7.77 (d, J = 8.4, 2H, Ar-H), 7.48

6

(

d, J = 8.0, 2H, Ar-H), 6.15 (s, 2H, NH ), 5.82 (s, 2H, NH ), 4.77 (d, J = 4.8, 1H, CHOH), 4.55 (t, J = 5.6,

2 2

1

H, CH OH), 4.14–4.06 (m, 2H, OCH ), 3.63–3.57 (m, 1H, CH), 3.31–3.25 (m, 2H, OCH ); ES-MS 302.1

2

+

(M + H) ; HRMS Calcd. for C H N O 302.1253, found 302.1250.

14 16 5 3

(

8

1

2

4

3

R)-2,4-Diamino-5-(4-methoxyphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10j). White solid 0.37 g, yield

◦

−1

4%; m.p. 188.4–188.9 C; IR: υmax/cm 3544 (OH), 3497 (OH), 3466 (NH), 3339 (NH), 1639 (C=N),

1

621 (C=N), 1591 (C=C), 1557 (C=C); H-NMR (DMSO-d )

δ

7.16 (d, J = 8.8, 2H, Ar-H), 6.92 (d, J = 8.8,

6

H, Ar-H), 5.94 (s, 2H, NH ), 5.46 (s, 2H, NH ), 4.73 (d, 1H, J = 4.8, CHOH), 4.49 (t, J = 5.8, 1H, CH OH),

2

.11–4.04 (m, 2H, OCH ), 3.76 (s, 3H, OCH ), 3.62–3.55 (m, 1H, CH), 3.33–3.24 (m, 2H, OCH ); ES-MS

2

3

2

+

07.1 (M + H) ; HRMS Calcd. for C H N O 307.1406, found 307.1404.

14

19

4

(

R)-2,4-Diamino-5-(3,4,5-trimethoxyphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10k). White solid 0.34 g,

◦

−1

yield 75%; m.p. 198.6–199.7 C; IR: υmax/cm 3489 (OH), 3439 (OH), 3356 (NH), 3208 (NH), 1626

(

1

C=N), 1589 (C=C), 1569 (C=C), 1489 (C=C); H-NMR (DMSO-d )

δ

6.55 (s, 2H, Ar-H), 5.96 (s, 2H,

6

NH ), 5.69 (s, 2H, NH ), 4.76 (d, J = 5.2, 1H, CHOH), 4.53 (t, J = 5.6, 1H, CH OH), 4.16–4.05 (m, 2H,

2

OCH ), 3.75 (s, 6H, OCH

ES-MS 367.2 (M + H) ; HRMS Calcd. for C H N O 367.1618, found 367.1616.

×

2), 3.68 (s, 3H, OCH ), 3.67–3.60 (m, 1H, CH), 3.38–3.28 (m, 2H, OCH );

2

3

2

+

16

23

4

6

(

R)-2,4-Diamino-5-[3-(2,2,2-triﬂuoroethoxymethyl)phenyl]-6-(1,2-dihydroxypropyl)pyrimidine

(

10l).

◦

−1

White solid 0.41 g, yield 82%; m.p. 48.2–49.3 C; IR: υmax/cm 3478 (OH), 3347 (NH), 3215 (NH),

1

612 (C=N), 1562 (C=C), 1492 (C=C), 1348(C-F); H-NMR (DMSO-d )

δ

7.36 (t, J = 7.8, 1H, Ar-H),

6

7

.20–7.23 (m,3H, Ar-H), 6.02 (s, 2H, NH ), 5.59 (s, 2H, NH ), 4.74 (d, J = 5.2, 1H, CHOH), 4.65 (s, 2H,

2 2

CH ), 4.51 (t, J = 5.6, 1H, CH OH), 4.09 (dd, J = 18.8 , J = 9.6 , 2H, CF CH ), 4.08 (d, J = 5.2, 2H,

2

1

2

3

2

+

OCH ), 3.62–3.55 (m, 1H, CH), 3.34–3.23 (m, 2H, OCH ); ES-MS 389.1(M + H) ; HRMS Calcd. for

C H F N O 389.1437, found 389.1435.

2

+

16

20

3

4

(

R)-2,4-Diamino-5-[3,5-dimethyl-4-(N-methoxyaminosulfonyl)phenyl]-6-(1,2-dihydroxypropyl)pyrimidine

◦

−1

10m). White solid 0.40 g, yield 88%; m.p. 98.3–99.3 C; IR: υmax/cm 3475 (OH), 3378 (NH), 3216

1

NH), 1610 (C=N), 1594 (C=C), 1562 (C=C), 1482(C=C); H-NMR (DMSO-d )

δ 10.29 (s, 1H, NH), 7.18

6

s, 2H, Ar-H), 6.12 (s, 2H, NH ), 5.80 (s, 2H, NH ), 4.79 (d, J = 4.8, 1H, CHOH), 4.56 (t, J = 5.8, 1H,

2

CH OH), 4.15–4.07 (m, 2H, OCH ), 3.64–3.58 (m, 1H, CH and OCH ), 3.33–3.27 (m, 2H, OCH ), 2.59 (s,

2

3

2

+

6

H, CH ); ES-MS 414.1(M + H) ; HRMS Calcd. for C H N O S 414.1447, found 414.1450.

3 16 24 5 6

(

0

(

R)-2,4-Diamino-5-[4-(morpholine-4-carbonyl)phenyl]-6-(1,2-dihydroxypropyl)pyrimidine (10n). White solid

◦

−1

.53 g, yield 90%; m.p. 210.1–211.3 C; IR (KBr): υmax/cm 3446 (OH), 3343 (NH), 3220 (NH), 1630

1

C=N), 1606 (C=C), 1591 (C=C), 1569 (C=C), 1511 (C=C); H-NMR (DMSO-d )

δ

7.36 (d, J = 8.4, 2H,

6

Ar-H), 7.32 (d, J = 8.4, 2H, Ar-H), 6.04 (s, 2H, NH ), 5.69 (s, 2H, NH ), 4.77 (d, J = 5.2, 1H, CHOH),

2

4

.53 (t, J = 5.6, 1H, CH OH), 4.10 (d, J = 5.6, 2H, OCH ), 3.63–3.59 (m, 1H, CH), 3.76–3.42 (m, 8H,

2

+

CH × 4), 3.30–3.27 (m, 2H, OCH ); ES-MS 390.2 (M + H) ; HRMS Calcd. for C H N O 390.1777,

2

18 24

5

found 390.1774.

(

R)-2,4-Diamino-5-[4-(3-triﬂuoromethoxyanilinocarbonyl)phenyl]-6-(1,2-dihydroxypropyl)pyrimidine (10o).

◦

−1

White solid 0.46 g, yield 91%; m.p. 142.0–143.6 C; IR: υmax/cm 3346 (OH), 3228 (NH), 1656 (C=N),

1

632 (C=N), 1607 (C=C), 1549 (C=C), 1490 (C=C), 1349(C-F); H-NMR (DMSO-d )

δ

10.49 (s, 1H, NH),

6

Molecules 2017, 22, 1592

18 of 27

7

.98–7.96 (m, 3H, Ar-H), 7.80 (dd, J = 8.0, J = 1.2, 1H, Ar-H), 7.51–7.45 (m, 3H, Ar-H), 7.09 (dt, J = 8.4,

1 2 1

J = 1.2, 1H, Ar-H), 6.10 (s, 2H, NH ), 5.71 (s, 2H, NH ), 4.77 (d, J = 5.2, 1H, CHOH), 4.55 (t, J = 5.6, 1H,

2

CH OH), 4.12 (d, J = 5.2, 2H, OCH ), 3.65–3.59 (m, 1H, CH), 3.33–3.26 (m, 2H, OCH ); ES-MS 480.1

2

+

(

M + H) ; HRMS Calcd. for C H F N O 480.1495, found 480.1492.

21 21 3 5 5

(

R)-2,4-Diamino-5-(4-methoxycarbonylphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10p). White solid 0.40 g,

◦

−1

yield 90%; m.p. 186.7–188.2 C; IR: υmax/cm 3449 (OH), 3330 (NH), 3219 (NH), 1705 (C=O), 1631

(

1

C=N), 1592 (C=C), 1514 (C=C); H-NMR (DMSO-d )

δ

7.91 (d, J = 8.4, 2H, Ar-H), 7.44 (d, J = 8.4, 2H,

6

Ar-H), 6.10 (s, 2H, NH ), 5.73 (s, 2H, NH ), 4.75 (d, J = 4.8, 1H, CHOH), 4.53 (t, J = 5.8, 1H, CH OH),

2

4

3

.09 (d, J = 5.2, 2H, OCH ), 3.86 (s, 3H, OCH ), 3.63–3.55 (m, 1H, CH), 3.30–3.25 (m, 2H, OCH ); ES-MS

2 3 2

+

35.1 (M + H) ; HRMS Calcd. for C H N O 335.1355, found 335.1350.

15

19

4

5

(

R)-2,4-Diamino-5-(3-methoxycarbonylphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (10q). White solid 0.40 g,

◦

−1

yield 90%; m.p. 140.6–141.5 C; IR: υmax/cm 3457 (OH), 3336 (NH), 3224 (NH), 1705 (C=O), 1658

(

1

C=N), 1600 (C=C), 1560 (C=C), 1490 (C=C); H-NMR (DMSO-d )

δ

7.84 (t, J = 1.2, 1H, Ar-H), 7.81 (dt,

6

J = 7.6, J = 1.6, 1H, Ar-H), 7.54 (dt, J = 8.0, J = 1.6, 1H, Ar-H), 7.49 (t, J = 7.6, 1H, Ar-H), 6.06 (s, 2H,

1

2

1

2

NH ), 5.68 (s, 2H, NH ), 4.72 (d, J = 5.2, 1H, CHOH), 4.50 (t, J = 5.6, 1H, CH OH), 4.09 (d, J = 5.6, 2H,

2

+

OCH ), 3.85 (s, 3H, OCH ), 3.61–3.54 (m, 1H, CH), 3.30–3.24 (m, 2H, OCH ); ES-MS 335.1 (M + H) ;

HRMS Calcd. for C H N O 335.1355, found 335.1358.

2

3

2

+

15

19

4

5

(

S)-2,4-Diamino-5-(4-chlorophenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11a). White solid 0.36 g, yield 85%;

◦

−1

m.p. 169.8–169.9 C; IR: υmax/cm 3446 (OH), 3396 (OH), 3336 (NH), 3214 (NH), 1606 (C=N), 1589

(

1

C=C), 1569 (C=C), 1556 (C=C), 1503(C=C), 797 (C-Cl); H-NMR (DMSO-d )

δ

7.38 (d, J = 8.4, 2H,

6

Ar-H), 7.27 (d, J = 8.4, 2H, Ar-H), 6.03 (s, 2H, NH ), 5.65 (s, 2H, NH ), 4.74 (d, J = 5.2, 1H, CHOH), 4.51

2

(

t, J = 5.6, 1H, CH OH), 4.08 (d, J = 5.6, 2H, OCH ), 3.63–3.56 (m, 1H, CH), 3.31–3.23 (m, 2H, OCH );

2

+

ES-MS 311.1 (M + H) ; HRMS Calcd. for C H ClN O 311.0911, found 311.0908.

13

16

4

3

(

S)-2,4-Diamino-5-(4-ﬂuorophenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11b). White solid 0.42 g, yield 95%;

◦

−1

m.p. 111.7–113.7 C; IR: υmax/cm 3482 (OH), 3343 (NH), 3212 (NH), 1614 (C=N), 1593 (C=C), 1562

(

1

C=C), 1509 (C=C), 1398 (C-F); H-NMR (DMSO-d )

δ

7.27 (dd, J = 8.8, J = 6.0, 2H, Ar-H), 7.16 (t,

6

1

2

J = 8.8, 2H, Ar-H), 6.00 (s, 2H, NH ), 5.57 (s, 2H, NH ), 4.73 (d, J = 5.2, 1H, CHOH), 4.50 (t, J = 5.8, 1H,

2

CH OH), 4.08 (d, J = 5.2, 2H, OCH ), 3.62–3.55 (m, 1H, CH), 3.33–3.23 (m, 2H, OCH ); ES-MS 295.1

2

+

(

M + H) ; HRMS Calcd. for C H FN O 295.1206, found 295.1208.

13 16 4 3

(

8

1

S)-2,4-Diamino-5-(3,4-dichlorophenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11c). White solid 0.35 g, yield

◦

−1

7%; m.p. 160.6–160.8 C; IR: υmax/cm 3504 (OH), 3476 (OH), 3383 (NH), 3346 (NH), 1649 (C=N),

1

617 (C=N), 1592 (C=C), 1569 (C=C), 1491 (C=C), 793 (C-Cl); H-NMR (DMSO-d )

δ

7.56 (d, J = 8.0, 2H,

6

Ar-H), 7.47 (d, J = 2.0, 1H, Ar-H), 7.24 (dd, J = 8.0, J = 2.0, 1H, Ar-H), 6.08 (s, 2H, NH ), 5.81 (s, 2H,

1

2

NH ), 4.75 (d, 1H, J = 5.2, CHOH), 4.53 (t, J = 5.6, 1H, CH OH), 4.14–4.06 (m, 2H, OCH ), 3.63–3.57 (m,

2

+

1

H, CH), 3.31–3.25 (m, 2H, OCH ); ES-MS 345.1 (M + H) ; HRMS Calcd. for C H Cl N O 345.0521,

2 13 15 2 4 3

found 345.0523.

(

S)-2,4-Diamino-5-(3-chloro-4-ﬂuorophenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11d). White solid 0.37 g,

◦

−1

yield 90%; m.p. 157.7–158.1 C; IR: υmax/cm 3484 (OH), 3329 (NH), 3210 (NH), 1622 (C=N), 1605

(

1

C=C), 1590 (C=C), 1561 (C=C), 1347 (C-F), 799 (C-Cl); H-NMR (DMSO-d )

δ

7.40 (dd, J = 7.2, J₂= 2.0

,

6

1

H, Ar-H), 7.36 (t, J = 8.8, 1H, Ar-H), 7.25–7.21 (ddd, J = 8.8, J = 4.8, J = 2.0, 1H, Ar-H), 6.05 (s, 2H,

1 2 3

NH ), 5.74 (s, 2H, NH ), 4.74 (d, J = 5.2, 1H, CHOH), 4.52 (t, J = 5.6, 1H, CH OH), 4.13–4.06 (m, 2H,

2

+

OCH ), 3.63–3.56 (m, 1H, CH), 3.30–3.26 (m, 2H, OCH ); ES-MS 329.1(M + H) ; HRMS Calcd. for

C H ClFN O 329.0817, found 329.0821.

2

+

13

15

4

3

(

S)-2,4-Diamino-5-(4-triﬂuoromethylphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11e). White solid 0.37 g,

◦

−1

yield 83%; m.p. 170.8–171.5 C; IR: υmax/cm 3620 (OH), 3512 (OH), 3334 (NH), 3225 (NH), 1661

(

1

C=N), 1632 (C=N), 1598 (C=C), 1554 (C=C), 1493 (C=C), 1339 (C-F); H-NMR (DMSO-d )

δ

7.67 (d,

6

J = 8.0, 2H, Ar-H), 7.50 (d, J = 8.0, 2H, Ar-H), 6.10 (s, 2H, NH ), 5.77 (s, 2H, NH ), 4.75 (d, J = 5.2, 1H,

2

Molecules 2017, 22, 1592

19 of 27

CHOH), 4.53 (t, J = 5.6, 1H, CH OH), 4.15–4.07 (m, 2H, OCH ), 3.64–3.57 (m, 1H, CH), 3.31–3.26 (m,

2

+

2

H, OCH ); ES-MS 345.1 (M + H) ; HRMS Calcd. for C H F N O 345.1175, found 345.1180.

2 14 16 3 4 3

(

S)-2,4-Diamino-5-(3-triﬂuoromethylphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11f). White solid 0.37 g,

◦

−1

yield 83%; m.p. 144.2–144.3 C; IR: υmax/cm 3474 (OH), 3389 (OH), 3348 (NH), 3224 (NH), 1617

(

1

C=N), 1594 (C=C), 1564 (C=C), 1496 (C=C), 1336 (C-F); H-NMR (DMSO-d )

δ

7.59–7.55 (m, 4H, Ar-H),

6

.09 (s, 2H, NH ), 5.74 (s, 2H, NH ), 4.72 (d, J = 4.8, 1H, CHOH), 4.50 (t, J = 5.8, 1H, CH OH), 4.12 (d,

2

+

J = 5.2, 2H, OCH ), 3.63–3.54 (m, 1H, CH), 3.31–3.25 (m, 2H, OCH ); ES-MS 345.1 (M + H) ; HRMS

Calcd. for C H F N O 345.1175, found 345.1177.

2

+

14

16

3

4

3

(

S)-2,4-Diamino-5-(4-triﬂuoromethoxyphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11g). White solid 0.44 g,

◦

−1

yield 90%; m.p. 141.7–143.2 C; IR: υmax/cm 3345 (OH), 3216 (NH), 1614 (C=N), 1595 (C=C), 1561

(

1

C=C), 1349 (C-F); H-NMR (DMSO-d )

δ

7.39 (d, J = 8.4, 2H, Ar-H), 7.31 (d, J = 8.4, 2H, Ar-H), 6.05 (s,

6

2

H, NH ), 5.68 (s, 2H, NH ), 4.76 (d, J = 4.8, 1H, CHOH), 4.52 (t, J = 5.6, 1H, CH OH), 4.10 (d, J = 5.2,

2 2 2

+

H, OCH ), 3.64–3.57 (m, 1H, CH), 3.31–3.24 (m, 2H, OCH ); ES-MS 361.1 (M + H) ; HRMS Calcd. for

2

+

C H F N O 361.1124, found 361.1127.

14

16

3

4

(

S)-2,4-Diamino-5-(3-triﬂuoromethoxyphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11h). White solid 0.44 g,

◦

−1

yield 90%; m.p. 120.5–122.0 C; IR: υmax/cm 3621 (OH), 3340 (OH), 3224 (NH), 1656 (C=N), 1629

(

1

C=N), 1609 (C=C), 1563 (C=C), 1499 (C=C), 1354 (C-F); H-NMR (DMSO-d )

δ

7.46 (t, J = 7.8, 2H, Ar-H),

6

7

.31 (d, J = 7.8, 2H, Ar-H), 7.23 (s, 1H, Ar-H), 7.19 (d, J = 7.8, 1H, Ar-H), 6.09 (s, 2H, NH ), 5.73 (s, 2H,

2

NH ), 4.74 (d, J = 4.8, 1H, CHOH), 4.51 (t, J = 5.6, 1H, CH OH), 4.10 (d, J = 5.2, 2H, OCH ), 3.63–3.56 (m,

2

+

1

H, CH), 3.31–3.25 (m, 2H, OCH ); ES-MS 361.1 (M + H) ; HRMS Calcd. for C H F N O 361.1124,

2

14 16

3

4

found 361.1122.

(

S)-2,4-Diamino-5-(4-cyanophenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11i). White solid 0.38 g, yield 86%;

◦

−1

m.p. 201.6–203.0 C; IR: υmax/cm 3458 (OH), 3359 (NH), 2230 (CN), 1631 (C=N), 1604 (C=C) 1586

(

1

C=C), 1563 (C=C), 1550 (C=C); H-NMR (DMSO-d )

δ

7.77 (d, J = 8.8, 2H, Ar-H), 7.48 (d, J = 8.8, 2H,

6

Ar-H), 6.15 (s, 2H, NH ), 5.83 (s, 2H, NH ), 4.76 (d, J = 5.2, 1H, CHOH), 4.54 (t, J = 5.6, 1H, CH OH),

2

+

4

.14–4.07 (m, 2H, OCH ), 3.62–3.58 (m, 1H, CH), 3.31–3.3124 (m, 2H, OCH ); ES-MS 302.1 (M + H) ;

2

+

HRMS Calcd. for C H N O 302.1253, found 302.1251.

14

16

5

3

(

9

1

S)-2,4-Diamino-5-(4-methoxyphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11j). White solid 0.36 g, yield

◦

−1

1%; m.p. 187.4–188.7 C; IR: υmax/cm 3542 (OH), 3496 (OH), 3467 (NH), 3339 (NH), 1639 (C=N),

1

621 (C=N), 1608 (C=C), 1591 (C=C), 1558 (C=C); H-NMR (DMSO-d )

δ

7.15 (d, J = 8.8, 2H, Ar-H),

6

.92 (d, J = 8.8, 2H, Ar-H), 5.94 (s, 2H, NH ), 5.46 (s, 2H, NH ), 4.73 (d, J = 4.8, 1H, CHOH), 4.50 (t,

2

J = 5.6, 1H, CH OH), 4.11–4.04 (m, 2H, OCH ), 3.76 (s, 3H, OCH ), 3.62–3.56 (m, 1H, CH), 3.32–3.25 (m,

2

3

+

2

H, OCH ); ES-MS 307.1 (M + H) ; HRMS Calcd. for C H N O 307.1406, found 307.1408.

2 14 19 4 4

(

S)-2,4-Diamino-5-(3,4,5-trimethoxyphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11k). White solid 0.40 g,

◦

−1

yield 89%; m.p. 199.1–200.3 C; IR: υmax/cm 3489 (OH), 3439 (OH), 3356 (NH), 3208 (NH), 1626

(

1

C=N), 1589 (C=C), 1569 (C=C), 1489 (C=C); H-NMR (DMSO-d )

δ

6.55 (s, 2H, Ar-H), 5.96 (s, 2H,

6

NH ), 5.64 (s, 2H, NH ), 4.76 (d, J = 4.8, 1H, CHOH), 4.53 (t, J = 5.8, 1H, CH OH), 4.14–4.07 (m, 2H,

2

OCH ), 3.75 (s, 6H, OCH ), 3.68 (s, 3H, OCH ), 3.65–3.60 (m, 1H, CH), 3.38–3.28 (m, 2H, OCH ); ES-MS

2

3

2

+

3

67.2 (M + H) ; HRMS Calcd. for C H N O 367.1618, found 367.1620.

16 23 4 6

(

S)-2,4-Diamino-5-[3-(2,2,2-triﬂuoroethoxymethyl)phenyl]-6-(1,2-dihydroxypropyl)pyrimidine

(11l).

◦

−1

White solid 0.38 g, yield 84%; m.p. 42.7–43.5 C; IR: υmax/cm 3477 (OH), 3346 (NH), 3215 (NH), 1613

1

(

C=N), 1561 (C=C), 1495 (C=C), 1346 (C-F); H-NMR (DMSO-d )

δ

7.36 (t, J = 7.8, 1H, Ar-H), 7.23–7.20

6

m,3H, Ar-H), 6.01 (s, 2H, NH ), 5.57 (s, 2H, NH ), 4.73 (d, J = 5.2, 1H, CHOH), 4.66 (s, 2H, OCH ), 4.50

2

t, J = 5.6, 1H, CH OH), 4.13–4.06 (m, 4H, OCH and CF CH ), 3.62–3.55 (m, 1H, CH), 3.31–3.23 (m, 2H,

2

3

2

+

OCH ); ES-MS 389.1(M + H) ; HRMS Calcd. for C H F N O 389.1437, found 389.1440.

2

16 20

3

4

(

S)-2,4-Diamino-5-[3,5-dimethyl-4-(N-methoxyaminosulfonyl)phenyl]-6-(1,2-dihydroxypropyl)pyrimidine

◦ −1

11m). White solid 0.31 g, yield 85%; m.p. 93.0–93.8 C; IR: υmax/cm 3463 (OH), 3379 (NH), 3215

Molecules 2017, 22, 1592

20 of 27

1

(

NH), 1612 (C=N), 1594 (C=C), 1563 (C=C); H-NMR (DMSO-d )

δ

10.29 (s, 1H, NH), 7.18 (s, 2H, Ar-H),

6

.11 (s, 2H, NH ), 5.78 (s, 2H, NH ), 4.77 (d, J = 5.2, 1H, CHOH), 4.55 (t, J = 5.6, 1H, CH OH), 4.15–4.07

2

(

m, 2H, OCH ), 3.62–3.58 (m, 4H, OCH and CH), 3.32–3.28 (m, 2H, OCH ), 2.59 (s, 6H, CH

×

2);

2

3

2

3

+

ES-MS 414.1(M + H) ; HRMS Calcd. for C H N O S 414.1447, found 414.1449.

16

24

5

6

(

0

1

S)-2,4-Diamino-5-[4-(morpholino-4-carbonyl)phenyl]-6-(1,2-dihydroxypropyl)pyrimidine (11n). White solid

◦

−1

.35 g, yield 86%; m.p. 209.6–210.2 C; IR: υmax/cm 3443 (OH), 3342 (NH), 3220 (NH), 1629 (C=N),

1

609 (C=C), 1590 (C=C), 1568 (C=C), 1511 (C=C); H-NMR (DMSO-d )

δ

7.36 (d, J = 8.4, 2H, Ar-H), 7.34

6

(

d, J = 8.4, 2H, Ar-H), 6.04 (s, 2H, NH ), 5.69 (s, 2H, NH ), 4.77 (d, J = 5.2, 1H, CHOH), 4.53 (t, J = 5.6,

2 2

1

H, CH OH), 4.10 (d, J = 5.2, 2H, OCH ), 3.63–3.59 (m, 1H, CH), 3.74–3.38 (m, 8H, CH

2

×

4), 3.32–3.25

2

+

(m, 2H, OCH ); ES-MS 390.2 (M + H) ; HRMS Calcd. for C H N O 390.1777, found 390.1779.

2 18 24 5 5

(

S)-2,4-Diamino-5-[4-(3-triﬂuoromethoxyanilinocarbonyl)phenyl]-6-(1,2-dihydroxypropyl)pyrimidine (11o).

◦

−1

White soilid 0.44 g, 87%; m.p. 143.2–145.0 C; IR: υmax/cm 3336 (OH), 3225 (NH), 1658 (C=N), 1606

(

1

C=C), 1549 (C=C), 1491 (C=C), 1350 (C-F); H-NMR (DMSO-d )

δ

10.49 (s, 1H, NH), 7.98–7.96 ( m,

6

3

H, Ar-H), 7.80 (dd, J = 8.2, J = 1.0, 1H, Ar-H), 7.51–7.45 (m, 3H, Ar-H), 7.09 (dt, J = 8.4, J = 1.2, 1H,

1 2 1 2

Ar-H), 6.10 (s, 2H, NH ), 5.70 (s, 2H, NH ), 4.77 (d, J = 5.2, 1H, CHOH), 4.55 (t, J = 5.8, 1H, CH OH),

2

+

4

.12 (d, J = 5.2, 2H, CH ), 3.58–3.65 (m, 1H, CH), 3.28–3.33 (m, 2H, OH); ES-MS 480.1 (M + H) ; HRMS

2

+

Calcd. for C H F N O 480.1495, found 480.1497.

21

3

5

(

S)-2,4-Diamino-5-(4-methoxycarbonylphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11p). White solid 0.32 g,

◦

−1

yield 90%; m.p. 211.3–212.4 C; IR: υmax/cm 3562 (OH), 3450 (NH), 3227 (NH), 3217 (NH), 1712

(

1

C=O), 1629 (C=N), 1592 (C=C), 1573 (C=C), 1555 (C=C), 1515 (C=C); H-NMR (DMSO-d )

δ

7.92 (d,

6

J = 8.4, 2H, Ar-H), 7.44 (d, J = 8.4, 2H, Ar-H), 6.10 (s, 2H, NH ), 5.74 (s, 2H, NH ), 4.74 (d, J = 5.2,

2

1

H, CHOH), 4.52 (t, J = 5.6, 1H, CH OH), 4.13–4.06 (m, 2H, OCH ), 3.86 (s, 3H, OCH ) 3.6–3.57 (m,

2 2 3

+

1H, CH), 3.3–3.24 (m, 2H, OCH ); ES-MS 335.1 (M + H) ; HRMS Calcd. for C H N O 335.1355,

2 15 19 4 5

found 335.1357.

(

S)-2,4-Diamino-5-(3-methoxycarbonylphenyl)-6-(1,2-dihydroxypropyl)pyrimidine (11q). White solid 0.38 g,

◦

−1

yield 85%; m.p. 181.4–182.3 C; IR: υmax/cm 3577 (OH), 3525 (OH), 3453 (NH), 3338 (NH), 1705

(

1

C=O), 1637 (C=N), 1590 (C=C), 1558 (C=C), 1501 (C=C); H-NMR (DMSO-d )

δ

7.84 (s, 1H, Ar-H), 7.81

6

(

d, J = 7.2, 1H, Ar-H) 7.54 (d, J = 7.6, 1H, Ar-H), 7.49 (t, J = 7.6, 1H, Ar-H), 6.06 (s, 2H, NH ), 5.69 (s,

2

3

H, NH ), 4.72 (d, J = 4.8, 1H, CHOH), 4.50 (t, J = 5.8, 1H, CH OH), 4.09 (d, J = 5.2, 2H, OCH ), 3.85 (s,

2 2 2

+

H, OCH ) 3.61–3.54 (m, 1H, CH), 3.30–3.22 (m, 2H, OCH ); ES-MS 335.1 (M + H) ; HRMS Calcd. for

3

2

+

C H N O 335.1355, found 335.1353.

15

19

4

5

General Procedure for the Synthesis of 2,4-Diamino-6-substituted Pyrimidine Derivatives 13a-d

Under argon, to a solution of substituted methanol 12a-d (17.45 mmol) in dry DMSO or

THF (30 mL) was added NaH (60%, 21.82 mmol) and stirred at room temperature for 1 h.

2,4-Diamino-6-chloropyrimidine (2) (8.73 mmol) was added and stirred at room temperature for 11 h.

The reaction solution was quenched with sat NH Cl (50 mL) and extracted with EtOAc (150 mL × 3),

4

and the combined organic layers dried with Na SO , ﬁltered and concentrated. The residue was

2

4

puriﬁed by column chromatography on silica gel using CH Cl /CH OH (50:1, v/v) as the eluting

2

3

solvent to give compounds 13a-d.

◦

2

,4-Diamino-6-(2-methoxyethoxy)pyrimidine (13a). White solid 1.04 g, yield 65%. m.p. 152.9–153.9 C; IR:

−

1

υmax/cm 3440 (NH), 3368 (NH), 1636 (C=N), 1563 (C=C), 1118 (C-O-C); H-NMR (DMSO-d )

δ

6.00

6

(

s, 2H, NH ), 5.85 (s, 2H, NH ), 5.02 (s, 1H, Ar-H), 4.23–4.17 (m, 2H, CH ), 3.59–3.51 (m, 2H, CH ), 3.27

2 2 2 2

+

s, 3H, CH ); ES-MS 185.1 (M + H) ; HRMS Calcd. for C H ClN O 185.1039, found 185.1040.

3

16 20

4

3

◦

2

,4-Diamino-6-(3-methoxylpropoxy)pyrimidine (13b). White solid 1.07 g, yield 62%. m.p. 104.2–104.8 C;

−

1

IR: υmax/cm 3456 (NH), 3343 (NH), 1656 (C=N), 1572 (C=C), 1207 (C-O-C); H-NMR (DMSO-d )

6

δ

5.98 (s, 2H, NH ), 5.84 (s, 2H, NH ), 5.02 (s, 1H, Ar-H), 4.11 (t, J = 6.6, 2H, CH ), 3.40 (t, J = 6.3, 2H,

2 2 2

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+

CH ), 3.23 (s, 3H, CH ), 1.84 (m, 2H, CH ); ES-MS 199.1 (M + H) ; HRMS Calcd. for C H ClN O

2

3

2

16 20

4

3

1

99.1195, found 199.1198.

◦

2

,4-Diamino-6-(thiazole-5-methoxy)pyrimidine (13c). Yellow solid 1.44 g, yield 74%. m.p. 156.8–161.3 C;

−

1

IR: υmax/cm 3466 (NH), 3320 (NH), 1625 (C=N), 1570 (C=C), 1454 (C=C), 1138 (C-O-C), 1061 (C-O-C);

δ 9.06 (s, 1H, Ar-H), 7.97 (s, 1H, Ar-H), 6.07 (s, 2H, NH ), 5.98 (s, 2H, NH ), 5.44

2 2

s, 2H, CH ), 5.04 (s, 1H, Ar-H); ES-MS 224.0 (M + H) ; HRMS Calcd. for C H ClN O 224.0606,

1

H-NMR (DMSO-d₆)

+

(

2 16 20 4 3

found 224.0608.

2

,4-Diamino-6-(1-benzyl-1H-1,2,3-triazole-4-methoxy)pyrimidine (13d). White solid 2.05 g, yield 79%.

◦

−1

m.p.131.4–135.7 C; IR: υmax/cm 3421 (NH), 3333 (NH), 1631 (C=N), 1588 (N=N), 1451 (C=C), 1422

1

(

C=C), 1197 (C-O-C); H-NMR (DMSO-d )

δ

8.20 (s, 1H, Ar-H), 7.36 (m, 5H, Ar-H), 6.04 (s, 2H, NH ),

6

2

+

5

.96 (s, 2H, NH ), 5.59 (s, 2H, CH ), 5.20 (s, 2H, CH ), 5.02 (s, 1H, Ar-H); ES-MS 298.1 (M + H) ; HRMS

2

+

Calcd. for C H ClN O 298.1416, found 298.1412.

16

20

4

3

General Procedure for the Synthesis of 2,4-Diamino-5-iodine-6-substitutedpyrimidine Derivatives

4a-d

1

Under argon, to a solution of 13-d (6.06 mmol) in dry CH CN (35 mL) was added

3

N-iodosuccinimide (9.09 mmol) and stirred at room temperature for 12 h. The reaction solution

was extracted with EtOAc (150 mL 3), and the combined organic layers were washed by NaHSO₃,

sat. NaHCO and sat. NaCl, and dried with Na SO , ﬁltered and concentrated. The residue was

×

3

2

4

puriﬁed by column chromatography on silica gel using CH Cl /CH OH (80:1, v/v) as the eluting

2

3

solvent to give compounds 14a-d.

◦

2

,4-Diamino-5-iodo-6-(2-methoxyethoxy)pyrimidine (14a). Yellow solid 1.50 g, 80%. m.p. 109.7–111.0 C;

−

1

IR: υmax/cm 3424 (NH), 3345 (NH), 1705 (C=N), 1554 (C=C), 1116 (C-O-C), 523 (C-I); H-NMR

DMSO-d₆) 6.10 (s, 4H, NH ), 4.31–4.27 (m, 2H, CH ), 3.61–3.57 (m, 2H, CH ), 3.31 (s, 3H, CH );

ES-MS 310.9 (M + H) ; HRMS Calcd. for C H ClN O 311.0005, found 311.0003.

(

δ

2

3

+

16

20

4

3

◦

,4-Diamino-5-iodo-6-(3-methoxypropoxy)pyrimidine (14b). Yellow solid 1.37 g, 70%. m.p. 124.0–125.8 C;

2

−

1

IR: υmax/cm 3472 (NH), 3357 (NH), 1631 (C=N), 1553 (C=C), 1145 (C-O-C), 532 (C-I); H-NMR

DMSO-d₆) 6.09 (s, 4H, NH ), 4.20 (t, J = 6.4, 2H, CH ), 3.45 (t, J = 6.3, 2H, CH ), 3.24 (s, 3H, CH ),

(

1

δ

2

3

+

.86 (m, 2H, CH ); ES-MS 325.0 (M + H) ; HRMS Calcd. for C H ClN O 325.0161, found 325.0159.

2 16 20 4 3

2

,4-Diamino-5-iodo-6-(thiazole-5-methoxy)pyrimidine (14c). Yellow solid 1.90 g, yield 90%. m.p.

◦

−1

178.0–179.9 C; IR: υmax/cm 3436 (NH), 3305 (NH), 1624 (C=N), 1541 (C=C), 1442 (C=C), 1127

1

(C-O-C), 1103 (C-O-C), 545 (C-I); H-NMR (DMSO-d )

δ

9.08 (d, J = 0.5, 1H, Ar-H), 8.01 (d, J = 0.6, 1H,

6

+

Ar-H), 6.24 (s, 4H, NH ), 5.51 (s, 2H, CH ); ES-MS 349.9 (M + H) ; HRMS Calcd. for C H ClN O

2

16 20

4

3

49.9572, found 349.9572.

2

9

,4-Diamino-5-iodo-6-(1-benzyl-1H-1,2,3-triazole-4-methoxy)pyrimidine (14d). Yellow solid 2.38 g, yield

◦

−1

3%. m.p. 156.4–159.1 C; IR: υmax/cm 3441 (NH), 3340 (NH), 1618 (C=N), 1552 (N=N), 1446 (C=C),

1

1280 (C-O-C); H-NMR (DMSO-d )

δ

8.51 (s, 1H, Ar-H), 7.86–7.74 (m, 5H, Ar-H), 6.28 (s, 2H, NH ),

6

2

+

6

.20 (s, 2H, NH ), 6.08 (s, 2H, CH ), 5.81 (s, 2H, CH ); ES-MS 424.0 (M + H) ; HRMS Calcd. for

2

+

C H ClN O 424.0383, found 424.0380.

16

20

4

3

General Procedure for the Synthesis of 2,4-Diamino-5-aryl-6-substitutedpyrimidine Derivatives 16a-p

A) Under argon, to a mixed solution of EtOH/toluene/H O (1:2:1, 60 mL) was added compounds

(

2

1

4a-d (2.36 mmol), substituted phenylboronic acid 15 (3.55 mmol), Pd(dbpf)Cl (0.02 mmol) and K CO

2 2 3

◦

(

3.55 mmol) consecutively and then stirred at 90 C for 24 h. The reaction solution was extracted with

EtOAc (150 mL 3), and the combined organic layers were washed by H O and dried with Na SO ,

×

2

4

ﬁltered and concentrated. The residue was puriﬁed by column chromatography on silica gel using

CH Cl /CH OH (80:1, v/v) as the eluting solvent to the desired compounds.

2

3

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(

B) In a pressure tube, to a mixed solution of THF/H O (1:1, 50 mL) was added compounds 14a-d

2

(

2.40 mmol), substituted phenylboronic acid 15 (3.60 mmol), Pd(dbpf)Cl (0.03 mmol) and K CO

2 2 3

◦

3.60 mmol) consecutively and then stirred at 70 C for 20 h. The reaction solution was extracted with

3), and the combined organic layers were washed by H O and dried with Na SO ,

EtOAc (150 mL

×

2

4

ﬁltered and concentrated. The residue was puriﬁed by column chromatography on silica gel using

CH Cl /CH OH (60:1, v/v) as the eluting solvent to the desired compounds.

2

3

2

0

,4-Diamino-5-(4-triﬂuoromethoxyphenyl)-6-(2-methoxyethoxy)pyrimidine (16a). Method A. White solid

◦

−1

.60 g, yield 74%. m.p. 178.3–179.0 C; IR: υmax/cm 3477 (NH), 3349 (NH), 1619 (C=N), 1553 (C=C),

1

4

3

486 (C=C), 1447 (C=C), 1275 (C-F), 1222 (C-O-C), 1143 (C-O-C); H-NMR (DMSO-d )

δ

7.38–7.29 (m,

6

H, Ar-H), 6.04 (s, 2H, NH ), 5.67 (s, 2H, NH ), 4.28–4.23 (m, 2H, CH ), 3.52–3.46 (m, 2H, CH ), 3.18 (s,

2

+

H, CH ); ES-MS 345.1 (M + H) ; HRMS Calcd. for C H ClN O 345.1175, found 345.1176.

3

16 20

4

3

2

0

,4-Diamino-5-(3-triﬂuoromethoxyphenyl)-6-(2-methoxyethoxy)pyrimidine (16b). Method A. White solid

◦

−1

.60 g, yield 74%. m.p. 80.1–81.7 C; IR: υmax/cm 3347 (NH), 3393 (NH), 1620 (C=N), 1561 (C=C),

1

1451 (C=C), 1292 (C-F), 1207 (C-O-C), 1157 (C-O-C); H-NMR (DMSO-d ) 7.49–7.44 (m, 1H, Ar-H),

6

7

.29 (d, J = 7.8, 1H, Ar-H), 7.20 (d, J = 6.4, 2H, Ar-H), 6.08 (s, 2H, NH ), 5.73 (s, 2H, NH ), 4.28–4.23

2 2

+

(m, 2H, CH ), 3.51–3.46 (m, 2H, CH ), 3.18 (s, 3H, CH ); ES-MS 345.1 (M + H) ; HRMS Calcd. for

2 2 3

+

C H ClN O 345.1175, found 345.1177.

16

20

4

3

2

,4-Diamino-5-[3-(2,2,2-triﬂuoroethoxymethyl)phenyl]-6-(2-methoxyethoxy)pyrimidine (16c). Method A.

−

1

Colorless oil 0.61 g, yield 70%; IR: υmax/cm 3480 (NH), 3383 (NH), 1607 (C=N), 1558 (C=C), 1437

1

(

C=C), 1279 (C-F), 1160 (C-O-C), 1121 (C-O-C); H-NMR (DMSO-d )

δ

7.40–7.34 (m, 1H, Ar-H), 7.25–7.17

6

(

m, 3H, Ar-H), 6.01 (s, 2H, NH ), 5.57 (s, 2H, NH ), 4.65 (s, 2H, CH ), 4.27–4.22 (m, 2H, CH ), 4.09 (q,

2

+

J = 9.4, 2H, CH ), 3.51–3.45 (m, 2H, CH ), 3.18 (s, 3H, CH ); ES-MS 373.1 (M + H) ; HRMS Calcd. for

C H ClN O 373.1488, found 373.1486.

2

+

2

3

16

20

4

3

2

,4-Diamino-5-[3,5-dimethyl-4-(N-methoxyaminosulfonyl)phenyl]-6-(2-methoxyethoxy)pyrimidine (16d).

◦

−1

Method A. White solid 0.68 g, yield 73%. m.p. 167.3–171.4 C; IR: υmax/cm 3466 (NH), 3364

1

(

NH), 1633 (C=N), 1587 (C=C), 1507 (NH), 1435 (C=C), 1157 (SO ), 1078 (SO ), 1014 (SO ); H-NMR

2

(

DMSO-d₆)

δ 10.26 (s, 1H, NH), 7.15 (s, 2H, Ar-H), 6.09 (s, 2H, NH ), 5.77 (s, 2H, NH ), 4.29–4.23 (m,

2 2

2

H, CH ), 3.62 (s, 3H, CH ), 3.53–3.47 (m, 2H, CH ), 3.22 (s, 3H, CH ), 2.59 (s, 6H, CH ); ES-MS 398.1

2

+

3

2

3

+

(M + H) ; HRMS Calcd. for C H ClN O 398.1498, found 398.1497.

16 20 4 3

2

,4-Diamino-5-[4-(3-triﬂuoromethoxyanilinoformoxyl)phenyl)-6-(2-methoxyethoxy)pyrimidine

(16e).

◦

−1

Method A. White solid 0.77 g, yield 70%. m.p. 96.50–100.1 C; IR: υmax/cm 3424 (NH), 3345 (NH),

1

1705 (C=N), 1554 (C=C), 1116 (C-O-C), 523 (C-I); H-NMR (DMSO-d )

δ

10.48 (d, J = 10.3, 1H, NH),

6

7.94 (dd, J = 17.0, 4.9, 4H, Ar-H), 7.49 (t, J = 8.2, 1H, Ar-H), 7.43 (d, J = 8.4, 2H, Ar-H), 7.09 (d, J = 8.3,

1

3

H, Ar-H), 6.09 (s, 2H, NH ), 5.70 (s, 2H, NH ), 4.31–4.25 (m, 2H, CH ), 3.53–3.48 (m, 2H, CH ), 3.21 (s,

2

+

H, CH ); ES-MS 464.1 (M + H) ; HRMS Calcd. for C H ClN O 464.4252, found 464.4250.

3

16 20

4

3

2

0

,4-Diamino-5-(4-triﬂuoromethoxyphenyl)-6-(3-methoxypropoxy)pyrimidine (16f). Method A. White solid

◦

−1

.63 g, yield 75%. m.p. 189.3–190.9 C; IR: υmax/cm 3471 (NH), 3365 (NH), 1632 (C=N), 1557 (C=C),

1

451 (C=C), 1276 (C-F), 1207 (C-O-C), 1160 (C-O-C); H-NMR (DMSO-d )

δ

7.33 (m, 4H, Ar-H), 6.02 (s,

6

2

H, NH ), 5.64 (s, 2H, NH ), 4.15 (t, J = 6.5, 2H, CH ), 3.27 (t, J = 6.3, 2H, CH ), 3.16 (s, 3H, CH ), 1.74

2

3

+

(m, 2H, CH ); ES-MS 359.1 (M + H) ; HRMS Calcd. for C H ClN O 359.1331, found 359.1333.

2 16 20 4 3

2

0

,4-Diamino-5-(3-triﬂuoromethoxyphenyl)-6-(3-methoxypropoxy)pyrimidine (16g). Method A. White solid

◦

−1

.79 g, yield 94%. m.p. 165.1–165.4 C; IR: υmax/cm 3470 (NH), 3364 (NH), 1633 (C=N), 1559 (C=C),

1

1454 (C=C), 1283 (C-F), 1205 (C-O-C), 1154 (C-O-C); H-NMR (DMSO-d )

δ

7.47 (t, J = 8.0, 1H, Ar-H),

6

7

.27 (d, J = 7.8, 1H, Ar-H), 7.21 (dd, J = 8.3, 1.0, 1H, Ar-H), 7.17 (s, 1H, Ar-H), 6.06 (s, 2H, NH ), 5.70

2

(

s, 2H, NH ), 4.15 (t, J = 6.4, 2H, CH ), 3.29 (t, J = 6.3, 2H, CH ), 3.17 (s, 3H, CH ), 1.74 (m, 2H, CH );

2 2 2 3 2

+

ES-MS 359.1 (M + H) ; HRMS Calcd. for C H ClN O 359.1331, found 359.1330.

16

20

4

3

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2

0

,4-Diamino-5-(4-triﬂuoromethoxyphenyl)-6-(thiazole-5-methoxy)pyrimidine (16h). Method A. Yellow solid

◦

−1

.79 g, yield 87%. m.p. 188.3–189.6 C; IR: υmax/cm 3429 (NH), 3327 (NH), 1601 (C=N), 1556 (C=C),

1

1449 (C=C), 1245 (C-F), 1105 (C-O-C), 1025 (C-O-C); H-NMR (DMSO-d )

δ

9.03 (s, 1H, Ar-H), 7.91

6

(

s, 1H, Ar-H), 7.30 (s, 4H, Ar-H), 6.16 (s, 2H, NH ), 5.75 (s, 2H, NH ), 5.45 (s, 2H, CH ); ES-MS 384.0

2 2 2

+

M + H) ; HRMS Calcd. for C H ClN O 384.0742, found 384.0745.

16

20

4

3

2

0

,4-Diamino-5-(3-triﬂuoromethoxyphenyl)-6-(thiazole-5-methoxy)pyrimidine (16i). Method A. Yellow solid

◦

−1

.66 g, yield 73%. m.p. 142.8–143.2 C; IR (KBr): υmax/cm 3467 (NH), 3361 (NH), 1628 (C=N),

1

1556 (C=C), 1452 (C=C), 1261 (C-F), 1106 (C-O-C), 1042 (C-O-C); H-NMR (DMSO-d )

δ

9.01 (s, 1H,

6

Ar-H), 7.87 (s, 1H, Ar-H), 7.50–7.45 (m, 1H, Ar-H), 7.30 (d, J = 7.8, 1H, Ar-H), 7.21 (d, J = 6.4, 2H,

+

Ar-H), 6.20 (s, 2H, NH ), 5.79 (s, 2H, NH ), 5.45 (s, 2H, CH ); ES-MS 384.0 (M + H) ; HRMS Calcd. for

2

+

C H ClN O 384.0742, found 384.0740.

16

20

4

3

2

,4-Diamino-5-[3-(2,2,2-triﬂuoroethoxymethyl)phenyl]-6-(thiazole-5-methoxy)pyrimidine (16j). Method A.

◦

−1

Yellow solid 0.88 g, yield 91%. m.p. 126.3–129.0 C; IR: υmax/cm 3478 (NH), 3316 (NH), 1624 (C=N),

1

553 (C=C), 1442 (C=C), 1282 (C-F), 1158 (C-O-C); H-NMR (DMSO-d )

δ

9.02 (s, 1H, Ar-H), 7.90 (s, 1H,

6

Ar-H), 7.35 (t, J = 7.6, 1H, Ar-H), 7.21 (d, J = 7.7 , 1H, Ar-H), 7.14 (d, J = 8.6, 2H, Ar-H), 6.12 (s, 2H, NH ),

2

+

5

.64 (s, 2H, NH ), 5.44 (s, 2H, CH ), 4.64 (s, 2H, CH ), 4.08 (q, J = 9.4, 2H, CH ); ES-MS 412.1 (M + H)

;

2

+

2

HRMS Calcd. for C H ClN O 412.1055, found 412.1056.

16

20

4

3

2

,4-Diamino-5-[3,5-dimethyl-4-(N-methoxyaminosulfonyl)phenyl]-6-(thiazole-5-methoxy)pyrimidine (16k).

◦

−1

Method A. White solid 0.74 g, yield 72%. m.p. 139.2–142.6 C; IR: υmax/cm 3463 (NH), 3358

1

(

NH), 1616 (C=N), 1589 (C=C), 1490 (C=C), 1448 (C=C), 1168 (SO ), 1076 (SO ), 1009 (SO ); H-NMR

2

(

DMSO-d₆)

δ

10.26 (s, 1H, NH), 9.05 (s, 1H, Ar-H), 7.92 (s, 1H, Ar-H), 7.07 (s, 2H, Ar-H), 6.21 (s, 2H,

+

NH ), 5.84 (s, 2H, NH ), 5.46 (s, 2H, CH ), 3.61 (s, 3H, CH ), 2.57 (s, 6H, CH ); ES-MS 437.1 (M + H) ;

HRMS Calcd. for C H ClN O 437.1066, found 437.1065.

2

3

+

16

20

4

3

2

,4-Diamino-5-[4-(3-triﬂuoromethoxyanilinocarbonyl)phenyl]-6-(thiazole-5-methoxy)pyrimidine

(16l).

◦

−1

Method A. Yellow solid 1.17 g, yield 99%. m.p. 180.9–182.9 C; IR: υmax/cm 3430 (NH), 3340

1

(

NH), 3200 (NH), 1654 (C=O), 1608 (C=N), 1551 (C=C), 1484 (C=C), 1442 (C=C), 1257 (C-F); H-NMR

DMSO-d₆)

δ 10.46 (s, 1H, NH), 9.04 (s, 1H, Ar-H), 7.94 (d, J = 8.6, 4H, Ar-H), 7.79 (d, J = 8.2, 1H, Ar-H),

7

.48 (t, J = 8.2, 1H, Ar-H), 7.36 (d, J = 8.4, 2H, Ar-H), 7.09 (d, J = 8.2, 1H, Ar-H), 6.20 (s, 2H, NH ), 5.77

2

+

(s, 2H, NH ), 5.47 (s, 2H, CH ); ES-MS 503.1 (M + H) ; HRMS Calcd. for C H ClN O 503.1113,

2 2 16 20 4 3

found 503.1111.

2

,4-Diamino-5-(4-triﬂuoromethoxyphenyl)-6-(1-benzyl-1H-1,2,3-triazole-4-methoxy)pyrimidine

(16m).

◦

−1

Method A. White solid 0.98 g, yield 91%. m.p.159.5–160.6 C; IR: υmax/cm 3489 (NH), 3371 (NH),

1

1608 (C=N), 1556 (N=N), 1492 (C=C), 1439 (C=C), 1261 (C-O-C), 1219 (C-F); H-NMR (DMSO-d )

δ

6

8

.15 (s,1H, Ar-H), 7.40–7.23 (m, 9H, Ar-H), 6.17 (s, 2H, NH ), 5.73 (s, 2H, NH ), 5.57 (s, 2H, CH ), 5.24

2 2 2

+

(s, 2H, CH ); ES-MS 458.1 (M + H) ; HRMS Calcd. for C H ClN O 458.1552, found 458.1555.

2 16 20 4 3

2

,4-Diamino-5-(3-triﬂuoromethoxyphenyl)-6-(1-benzyl-1H-1,2,3-triazole-4-methoxy)pyrimidine

(

16n).

◦

−1

Method A. White solid 0.98 g, yield 91%. m.p. 171.9–173.0 C; IR: υmax/cm 3495 (NH), 3376 (NH),

1

1607 (C=N), 1556 (N=N), 1491 (C=C), 1431 (C=C), 1278 (C-O-C), 1219 (C-F); H-NMR (DMSO-d )

δ

6

8

.15 (s, 1H, Ar-H), 7.40–7.23 (m, 9H, Ar-H), 6.17 (s, 2H, NH ), 5.73 (s, 2H, NH ), 5.57 (s, 2H, CH ), 5.24

2 2 2

+

(s, 2H, CH ); ES-MS 458.1 (M + H) ; HRMS Calcd. for C H ClN O 458.1552, found 458.1553.

2 16 20 4 3

2

,4-Diamino-5-(4-methoxycarbonylphenyl)-6-(1-benzyl-1H-1,2,3-triazole-4-methoxy)pyrimidine

(

16o).

◦

−1

Method B. Gray solid 0.70 g, yield 68%. m.p. 218.6–222.3 C; IR: υmax/cm 3487 (NH), 3440 (NH),

1

1709 (C=O), 1616 (C=N), 1555 (N=N), 1483 (C=C), 1440 (C=C), 1285 (C-O-C); H-NMR (DMSO-d ):

6

8

.16 (s, 1H, Ar-H), 7.87 (d, J = 8.4, 2H, Ar-H), 7.39–7.25 (m, 7H, Ar-H), 6.20 (s, 2H, NH ), 5.77 (s, 2H,

2

+

NH ), 5.57 (s, 2H, CH ), 5.24 (s, 2H, CH ), 3.86 (s, 3H, CH ); ES-MS 432.1 (M + H) ; HRMS Calcd. for

C H ClN O 432.1784, found 432.1786.

2

3

+

16

20

4

3

Molecules 2017, 22, 1592

24 of 27

2

,4-Diamino-5-(3-methoxycarbonylphenyl)-6-(1-benzyl-1H-1,2,3-triazole-4-methoxy)pyrimidine

(16p).

◦

−1

Method B. Yellow solid 0.53 g, yield 51%. m.p. 210.4–213.7 C; IR: υmax/cm 3432 (NH), 3351 (NH),

1

706 (C=O), 1619 (C=N), 1564 (N=N), 1485 (C=C), 1442 (C=C), 1252 (C-O-C); H-NMR (DMSO-d )

δ

6

8

.13 (s, 1H, Ar-H), 7.80 (dd, J = 8.8, 4.4, 2H, Ar-H), 7.44 (d, J = 5.1, 2H, Ar-H), 7.34 (dd, J = 9.1, 6.9, 3H,

Ar-H), 7.28–7.24 (m, 2H, Ar-H), 6.16 (s, 2H, NH ), 5.71 (s, 2H, NH ), 5.56 (s, 2H, CH ), 5.24 (s, 2H, CH ),

2

+

3

.82 (s, 3H, CH ); ES-MS 432.1 (M + H) ; HRMS Calcd. for C H ClN O 432.1784, found 432.1785.

3

16 20

4

3

.3. Determination of Anti-Mycobacterial Activity

The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC)

of the test compounds were determined as described previously [19] with minor modiﬁcations.

In brief, the MIC was determined using the microbroth dilution method in 96-well microtitre plates.

Mycobacterium tuberculosis H37Ra (ATCC 25177) was used as the test strain, at a ﬁnal inoculum of

5

approximately 10 cfu/mL. The compounds were dissolved in DMSO and diluted in 1% DMSO in 7H9

broth to obtain concentrations ranging from 100

sealed with Paraﬁlm and incubated at 36 C for 28 days. On days 14 and 28, all wells were observed

µ

g/mL to 1.56

µ

g/mL. All inoculated plates were

◦

for visible growth, and 10

µ

L was removed from each well for subculturing on compound-free 7H10

◦

agar plates which were incubated for six weeks at 36 C. For each compound, the lowest concentration

to inhibit the growth of H37Ra in broth culture was taken to be the MIC. The MBC was the lowest

concentration to inhibit the formation of colonies on the agar subculture, up to six weeks of incubation.

3

.4. Molecular Docking

The crystal structure of mt-DHFR in complex with NADPH and MTX (PDB ID: 1DF7) [20] was

used as the receptor for molecular docking, which was performed by GOLD Docking: GOLD (v 5.2.2

Genetic Optimization for Ligand Docking) [21]. The bound ligand, MTX was used as a reference to

indicate the binding site, and each molecule was docked 10 times with the default automatic genetic

algorithm parameter settings and the results were evaluated by Gold Score.

3

.5. Molecular Dynamic Simulation

The geometry of the docked ligand was optimized by using the B3LYP 6–31 G* basis set within

Gaussian09 [22], and the atom-centered point charges were calculated to ﬁt the electrostatic potential

using RESP [23 ]. The parameters of NADPH were obtained from AMBER parameter database (http:

/

/www.pharmacy.manchester.ac.uk/bryce/amber/, uploaded by U. Ryde).

The docked compound and mt-DHFR complex was explicitly solvated in a truncated octahedral

box of TIP3P model water (at least 12 Å from the complex to avoid periodic artifacts from occurring)

by using Amber 16 with the amber ff14SB force ﬁeld. The system charges were neutralized by

+

adding enough K ions by using the tleap module (AmberTools 16). The explicit solvent models

and the NPT ensemble (T = 300 K; P = 1 atm) were performed on all molecular dynamic simulations.

Periodic boundary conditions (PBC) and particle-mesh-Ewald method (PME) [24] were used to treat

long-range electrostatic effects, with the temperature coupled to an external bath using a weak coupling

algorithm [25]. The non-bonded interaction cutoff was set as 8 Å. The bond interactions involving

H-atoms were constrained by using the SHAKE algorithm. The time step necessary to solve the

Newton’s equations was chosen to be equal to 2 fs and the trajectory ﬁles were collected every 10 ps.

All trajectory analysis was performed with the Ptraj module in the AmberTools 16 and examined

visually using VMD software [26].

The whole system was ﬁrst optimized by energy minimization, followed by 110 ns molecular

dynamic simulation, including 10 ns equilibration and 100 ns production simulations. The last

80 ns (1000 snapshots, 80 ps intervals) stable, equilibrated trajectories of the production simulation

were taken for binding free energy calculations performed by using MM-PBSA [27

–29] (included in

AMBERTOOLS 16).

Molecules 2017, 22, 1592

25 of 27

The entropy was estimated by using the Normal Mode program [28

,

29] within the AMBER16

suite. Because the entropy calculations are computationally intensive, only 100 snapshots from the last

8

0 ns trajectories were used for the normal-mode analysis.

4

. Conclusions

In conclusion, in order to occupy the GOL binding site on mt-DHFR and maintain proper

hydrophobicities to allow the compounds to function in the Mtb whole cell assay, we designed

and synthesized three series of compounds which contain a hydrophobic side chain. Among them,

the compounds with a thiazole side chain signiﬁcantly inhibited the growth of Mtb, and the best

inhibition effect was observed on compound 16l. More interestingly, this compound showed selectivity

for Mtb over vero cells, which makes it potentially useful as a lead compound for future studies on

anti-TB drugs. Unfortunately, we are currently unable to conﬁrm 16l as an mt-DHFR inhibitor by

performing the binding assay on the pure mt-DHFR enzyme.

Sulfonamides were the ﬁrst anti-bacterial agents to be used for the treatment of tuberculosis [30].

They were subsequently replaced by the standard ﬁrst-line anti-TB drugs rifampicin, isoniazid,

ethambutol and pyrazinamide, which had greater anti-TB activity. With the increase of multidrug

resistant TB in the past two decades, and the therapeutic failures with standard anti-TB drugs,

respiratory physicians have to resort the treatment with other antibiotics and chemotherapeutic agents

including the trimethoprim-sulfamethoxazole combination [31]. While sulfamethoxazole competes

with para-aminobenzoic acid (PABA) for the enzyme dihydropteroate synthetase, trimethoprim directly

inhibits the dihydrofolate reductase for the reduction of dihydrofolic acid to tetrahydrofolic acid.

This combination was found to have in vitro activity for up to 98% of Mtb isolates [32]. However, both

drugs are bacteriostatic, and are associated with drug resistance. Sulfonamide resistance has been

reported to result from a point mutation or the acquisition of a plasmid which enables the synthesis

of a dihydropteroic synthetase that has poor afﬁnity for sulfonamides. Similarly, trimethoprim can

be rendered ineffective by plasmid- and transposon-mediated production of an altered dihydrofolate

reductase that has markedly reduced afﬁnity for the drug. In some studies, trimethoprim, when used

by itself, has been shown to have little activity on some strains of Mtb [33]. The new compounds

we synthesized appear to be bactericidal against Mtb, making it a better drug for TB therapy in

immunocompromised patients. Further studies are required to test for the frequency of mutation to

resistance among clinical strains of Mtb and whether the compounds can be used in combination with

other anti-TB drugs or antibiotics for synergistic activity or to retard the development of resistance.

Acknowledgments: This work was supported by National Natural Science Foundation of China (81773582)

given to H.W.; National Natural Science Foundation of China (81460538, 81660588) given to W.H.;

IPSR/RMC/UTARRF/2015-C1/NO3 awarded to Y.F.N. by Universiti Tunku Abdul Rahman (UTAR),

Kuala Lumpur, Malaysia.

Author Contributions: Wei Hong, Hao Wang and Yun Fong Ngeow conceived and designed the experiments;

Yifan Ouyang, Hao Yang, Peng Zhang, Yu Wang, Sargit Kaur, Xuanli Zhu, Zhe Wang, and Yutong Sun performed

the experiments; Wei Hong and Hao Wang analyzed the data; Wei Hong, Hao Wang and Yun Fong Ngeow wrote

the paper.

Conﬂicts of Interest: All authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds 10a-q, 11a-q and 16a-p are available from the authors.

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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access

article distributed under the terms and conditions of the Creative Commons Attribution

CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Synthesis of 2,4-diaminopyrimidine core-based derivatives and biological evaluation of their anti-tubercular activities

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