Synthesis of Aza-acyclic Nucleoside Libraries of Purine, Pyrimidine

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Synthesis of Aza-acyclic Nucleoside Libraries of Purine, Pyrimidine and 1,2,4–Triazole Vibha Pathak, Ashish Kumar Pathak, and Robert C. Reynolds ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00136 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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ACS Combinatorial Science

Synthesis of Aza-acyclic Nucleoside Libraries of Purine, Pyrimidine and 1,2,4–Triazole Vibha Pathak,1 Ashish K. Pathak1* and Robert C. Reynolds2* 1Chemistry

Department, Drug Discovery Division, Southern Research, 2000 Ninth Avenue South,

Birmingham, Alabama 35205, USA 2Department

of Medicine, Division of Hematology and Oncology, University of Alabama at

Birmingham, NP 2540 J, 1720 2nd Avenue South, Birmingham, AL 35294-3300, USA

ACS Paragon Plus Environment

ACS Combinatorial Science 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Under the aegis of the Pilot Scale Library Program of the NIH Roadmap Initiative, a new library of propan-1-amine containing aza acyclic nucleosides were designed and prepared, and we now report a diverse set of 157 purine, pyrimidine and 1,2,4-triazole-N-acetamide analogues. These new nucleoside analogues were prepared in a parallel high throughput solution-phase format. A set of diverse amines were reacted with several nucleobase N-propaldehydes utilizing reductive amination with sodium triacetoxyborohydride coupling to produce a small and diverse aza acyclic nucleoside library. All reactions were performed using 24 well reaction blocks and an automatic reagent-dispensing platform under inert atmosphere. Final targets were purified on an automated system using solid sample loading pre-packed cartridges and pre-packed silica gel columns. All compounds were characterized by NMR and HRMS, and were analyzed for purity by HPLC prior to submission to the Molecular Libraries Small Molecule Repository (MLSMR). Initial screening through the Molecular Libraries Probe Production Centers Network (MLPCN), demonstrated diverse and interesting biological activities. Base = OBn Base N

O H

5 Bases

R1R2NH

Base

NaBH(OAc)3

N

OMe N

N N

N H

N

157 Compounds O

N

N

HN N

N H

N

NH2

O N

N H

N H

N

NHCbz

NR1R2

SMe N

N

N N H

O

N H

KEYWORDS: purine, pyrimidine, 1,2,4–triazole, aza–acyclic nucleosides, solution–phase, diversity


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


INTRODUCTION Modern drug discovery and the discipline of chemical biology have been energized by the advent of

high throughput technologies. Chemical biology involves the application of chemical techniques, tools, and analyses, and chemical libraries generated via synthetic or natural product chemistry to the study and manipulation of biological systems. Grandly, it encompasses applications of comprehensive chemical space to probe and study the entirety of biological diversity space. Even with the ready availability of diverse, drug-like commercial libraries, novel molecular diversity that more completely covers biologically relevant chemical space will continue to play an essential role in interrogating newly elucidated biological processes. Numerous screening programs have existed for decades through the U.S. National Institutes of Health (NIH) targeting cancer, infectious diseases, neurological disorders etc. With the advent of higher throughput technologies, NIH screening programs evolved onto higher throughput platforms and began to take advantage of readily available drug like commercial libraries in the mid to late 1990s.1 The Pilot Scale Library (PSL) Program was implemented to specifically produce libraries based on biologically relevant scaffolds with greater chemical diversity than provided by commercial chemical library space.2 An essential component of this initiative is the availability of high quality general compound screening libraries as well as focused sets that are relevant as biological probes or potential drug leads.3 In order to identify more biologically/medicinally relevant collections, a variety of computational filters have been applied including drug likeness and chemical reactivity.4,5 Still researchers continue to examine and explore alternative chemical space with a focus on improving selection for biologically relevant compound libraries.3 With the launch of the PSL program, it was realized that, in spite of the thousands of essential biological processes throughout the biome that are nucleoside dependent, few nucleosides and derivations were represented in commercial libraries.

Furthermore, we were inspired by nature,



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specifically the natural product nucleoside antibiotics that are highly diverse and show a multitude of biological activities including anticancer, antiparasitic, antifungal and antimicrobial.6

In general, these

defense compounds are known to target specific biological processes in other microbes (see Figure 1), and they do not typically enter standard nucleoside biology (e.g. phosphorylation and RNA or DNA metabolism). NH2 N N

H2 N O

HOOC H2 N

OH OH

(+)-Sinefungin

Figure 1.

HO

NH

N N R

H N O

N

Me

HO

NH O COOH

O HO

O

O

OHC

O

OH

Dihydropacidamycin

H2 N

HN

N

O

O

O OH OH

Nikkomycin Bx

HN R2

H N

O

R1

NH H2 N

O

O

N

O

O

O OMe OH

Capuramycin

Examples of diverse biologically active natural nucleoside antibiotics and synthetic

derivations Hence, we were specifically interested in acyclic nucleoside structures since acyclic nucleosidebased scaffolds with an amine linkage were not well represented in commercial chemical space, including the MLSMR (http://www.ncbi.nlm.nih.gov/pcsubstance).

Acyclic nucleosides and their

respective phosphonates have shown potent antiviral and cytostatic activities as reviewed recently.7 Acyclovir is an excellent example of an acyclic nucleoside that shows potent activity against herpes simplex viruses (HSV), and the discovery of this drug set the stage for further development of other potent clinical antiviral drugs in this class including Ganciclovir, Penciclovir, Famciclovir, and Adefovir. Peptide nucleic acid monomers (PNAM) which are synthetic analogs of nucleic acids with flexible pseudo-peptide chains have been synthesized and tested in a wide range of biological assays as well as for antisense applications.8 Recently, methods to synthesize stereospecific acyclic nucleosides have also been reported - see the rhodium-catalyzed asymmetric N-selective intermolecular addition of purine derivatives to terminal allenes9 as well as rhodium-catalyzed highly regio- and enantioselective asymmetric allylation of pyrimidines with racemic allylic carbonates.10 An efficient route to construct ACS Paragon Plus Environment

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chiral acyclic purine nucleosides containing a hemiaminal ester moiety has also been reported via threecomponent dynamic kinetic resolution of purines, aldehydes, and acid anhydrides.11 Furthermore, robust and mild chemistry exists through reductive amination (Nucleoside Base-CH2CH2CHO plus commercial amine diversity R-NH2 – see Figure 2) allowing the facile preparation of a diverse Nucleoside Base-CH2CH2-NHR diversity set.

Base

Diversity Point Bases

N Diversity Point Linker Length

NR1R2

Diversity Point Amines

Figure 2: General representation of library structures. Figure 2 presents a generalized structure for the library prepared showing three potential diversity points for library expansion, the nucleoside base, the linker, and a diverse set of amines. In terms of the base, we used three readily available 6-substituted purines (6-OMe, 6-SMe, and 6-OBn), one pyrimidine (CBz protected cytidine) and a five membered heterocycle (triazole) that is similar to imidazole analogs seen in the antiviral agent ribavirin, and certain nucleoside antibiotics (e.g. the polyoxins – see Nikkomycin Bx in Figure 1). Beyond a variety of nucleoside bases (purines, a pyrimidine, and a related heterocyle – triazole), these particular base analogs were used to demonstrate chemistry one might see and use in preparing a larger set of these acyclic analogs. While yields varied, sufficient quantities of a variety of analogs were prepared with these diverse base representatives. Further examples should be readily accessible (e.g. DNA/RNA bases such as G, A, T) which were not prepared in the current studies. The CBz-protected nucleoside base C was chosen to demonstrate a typical protection/deprotection requirement (necessary for the reductive amination diversification step) for the exocyclic NH2 group that would also likely be required for both A and G analogs as well as to demonstrate that various N-protected/substituted bases can be used to further enhance diversity should ACS Paragon Plus Environment


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that be desired. Next, one specific linker length of three carbons was explored in this small set that was exemplary of typical acyclic nucleoside analogs. For example, see structures of Fluorowillardine (PubChem CID 126569), Discadenine (PubChem CID 53477639), Eritadenine (PubChem CID 159961), and Acyclovir (PubChem CID 2022) with linker lengths ranging from 3-4 carbons. Preparation of the Base-Linker-CHO was carried out via standard alkylation chemistry using 3-bromo-1,1-dimethoxypropane with modest yields ranging from 53% to 71%, demonstrating that sufficient quantities of reagents for library preparation can be achieved with readily available starting materials. Furthermore, this chemistry should readily work for linkers in the 2-4 carbon ranges (or others), and the appropriate, commercially available reagents. While we specifically demonstrate the preparation of a variety of acyclic nucleoside analogs with an amine linkage using only a three-carbon linker, the chemistry is readily adaptable to other simple acyclic linkers as well as potentially more complex linkages. Finally, using the Base-Linker-CHO starting materials, the third diversification point was implemented using robust reductive amination chemistry with a diverse set of commercially available amines (e.g. primary, secondary, benzylic, aromatic and heteroaromatic etc.) to prepare a small focused library or 157 acyclic nucleoside analogs bearing an amine linkage. 

RESULTS AND DISCUSSION This report details the high throughput solution-phase parallel preparation of a small library of 157

propan-1-amine analogs comprising 6-(benzyloxy)-9H-purine, 6-(methoxy)-9H-purine, 6-(methylthio)9H-purine, benzyl (2-oxo-1,2-dihydropyrimidin-4-yl)carbamate, 6-amino-9H-purine and N-(3,4dimethylphenyl)-1H-1,2,4-triazole-3-carboxamide using automation. In terms of preparing a novel and diverse set, we first identified a commercial set of amines that were available in sufficient quantities and at a reasonable cost to feed into our synthetic program. These were further selected visually based on variety and expected reactivity in order to functionally feed into the coupling chemistry to supply a structurally diverse set of products. Finally, all expected products were drawn in ChemDraw and 3D ACS Paragon Plus Environment

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representations prepared to assess Tanimoto similarity with compounds already available in the MLSCN compound set. The Tanimoto similarity score is one accepted method of calculating the similarity between two compound fingerprints.12 The score is a value between 0 and 1, 0 indicating no similarity and 1 indicating 100% similarity. A Tanimoto score of 0.7 or above is representative of two molecules having high structural similarity. Each compound of the proposed set was compared with the complete MLSCN compound set (ca. 2008 - total 197,873 compounds). Of the current set supplied to the PSL application, only 9.3% had a Tanimoto score of 0.7 or above, meaning that the great majority of samples were significantly dissimilar relative to the MLSCN library at that time. In fact, 36 % of this set had Tanimoto scores of 0.5 or below, suggesting clear structural dissimilarity between the proposed compound set and the MLSCN library. Drug likeness relative to calculated parameters was also assessed. The predicted ranges of physicochemical properties such as LogD at pH 7.0, Log P, range of masses in amu, hydrogen bond acceptor (HBA), hydrogen bond donor (HBD) and total polar surface area (TPSA in Å2) of library members are represented in Table 1 and graphical representations of physicochemical properties distribution of library members are shown in Figure 3. The predicted values of physicochemical properties for these library members are, for the most part, well within accepted parameters for small molecule libraries. Table 1. Predicted values of physicochemical properties of library members Entry

Parameter

Range

Average

262–512

387

1

Molecular weight (MW)

2

LogP

-0.84–5.32

2.24

3

LogD at pH=7.40

-0.60–4.76

2.08

4

Number of H-bond donor (HBD)

0–3

1.5

5

Number of H-bond acceptors (HBA)

4–12

8.0

6

Total polar surface area (TPSA), Å2

59.31–168.73

114.02



15

15

10 5 0

20 % Frequency

20 % Frequency

20

10 5 0

Mass Ranges (amu)

-1.0 0.0 1.0 2.0 3.0 4.0 5.0 LogP

55 65 75 85 95 105115 TPSA (Å2)

60

30

% Frequency

% Frequency

0

5 0

40

10

10

LogD at pH=7.40

30 20

15

-4.00 -3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00

260 300 340 380 420 460 500

% Frequency

Figure 3. Graphical representations of physicochemical properties distribution of library members.

% Frequency

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20 10

40 20 0

0 3

4

5 6 HBA

7

8

9

0

1 2 HBD

3

4

Our first objective was to prepare the required starting materials in sufficient quantities to produce our libraries of purine, pyrimidine and 1,2,4-triazole-N-acetamide. The five nucleosidelike bases, 6-(benzyloxy)-9H-purine, 6-methoxy-9H-purine, 6-(methylthio)-9H-purine, benzyl (2oxo-1,2-dihydropyrimidin-4-yl)carbamate

and

N-(3,4-dimethylphenyl)-1H-1,2,4-triazole-3-

carboxamide, were acquired from commercial sources. In two steps from these reagents (Scheme 1), the N-propanals (1–5) were produced. Each base was reacted with 3-bromo-1,1-dimethoxypropane in the presence of K2CO3 using DMAc as a solvent at room temperature for 2 days to produce the desired intermediate acetals (1a–5a) in reasonable yields. Purification by column chromatography treatment with 1N HCl in dioxane for 4 hours at room temperature, and final chromatography gave clean products (1–5) in excellent yields for further library generation through reductive amination. Scheme 1. General synthetic approach for synthesis of compounds


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Base

BrCH2CH2CH(OCH3)2 K2CO3, DMAc, rt 2 days

Base

1N HCl

N

Dioxane rt, 4 h

O

1a-5a

R1R2NH

Base N

Base

STAB/DCM

O

N

H

O

6-162

1-5

NR1R2

O

Base =

N

N N

SMe

OMe

OBn

N 1

N

N N

N

HN N

N N

1

N 1

N O

O

O

H N

N N 1

N N 1

Aldehydes (1–5) were screened in several model reductive amination reactions for optimal adaptation to a 24–well reaction block parallel synthetic platform in 80–150 mg scale using a robotic reagent dispensing system. Sodium triacetoxyborohydride (STAB) in dichloromethane (DCM) was found to be the most suitable reagent to carry out library generation in a solutionphase parallel format. A 24–well MiniBlock XT solution–phase reaction block was used to carry out reactions with 17 x 110 mm glass reaction vessels as this system was compatible with Tecan automatic liquid handler (dispensing, retraction and aspiration) and a Genevac centrifuge solvent evaporator with adapters for Miniblocks.

In general, 3.0 mL of stock solution in dry

dichloromethane containing 80 mg of the starting aldehydes (1–3 or 5), or 4 mL of stock solution in DCM containing 150 mg of starting aldehyde (4) was dispensed into each vessel. Next, the appropriate amine (1.5 equivalents) as a 1M stock solution in DCM was added followed by a 1M STAB stock solution also in DCM containing 1.5 equivalents. All additions were under argon atmosphere. The reaction block was then loaded on a mechanical shaker and shaken at room temperature for 4 hours. After removal of solvent using a Genevac system, residues were dissolved in 2–3 mL of 15% CHCl3–MeOH and thin layer chromatography (TLC) was performed to check reaction progress. Reaction wells that did not show clean and efficient product formation were discarded and are not reported here. All reactions were performed once and reported yields are



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not optimized. The structures of analogs of 6-(benzyloxy)-9H-purine (6–30), 6-methoxy-9Hpurine (31–55), 6-(methylthio)-9H-purine (56–88), benzyl (2-oxo-1,2-dihydropyrimidin-4yl)carbamate (89–122) and N-(3,4-dimethylphenyl)-1H-1,2,4-triazole-3-carboxamide (123–147) are shown in Schemes 2 through 6 respectively.

In the case of the benzyl (2-oxo-1,2-

dihydropyrimidin-4-yl)carbamates (99, 104, 109, 110, 113, 114, 118, 119, 121 and 122) where sufficient quantities were available, 40 mg of these compounds were further reacted overnight with 40% NaOH solution in water (0.5 mL) and 2 mL MeOH at room temperature to produce 4-amino1-(3-aminopropyl)pyrimidin-2(1H)-one analogs (148–157) as shown in Scheme 7.

Certain

reductive amination reactions only produced disubstituted amines as the sole product (158–162) and these are shown in Figure 4. Each reaction mixture was aspirated via syringe, and filtered through a microfilter adapter onto a RediSep® solid sample loaded prepacked cartridge (2.5 g Silica) followed by drying under vacuum before purification on an automated medium pressure liquid chromatographic (MPLC) system using prepacked Silica RediSep Gold® columns. Each purified product was characterized by 1HNMR and high resolution mass spectra (HRMS) followed by purity assessment using HPLC. General experimental procedures, detailed analytical data and a complete list of structures along with their PubChem ID’s as hyperlinks to all library members are provided in Supporting Information.


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Scheme 2. Synthesis of 3-(6-(benzyloxy)-9H-purin-9-yl)propan-1-amine analogs (6–30) OCH3

1. Br

O N N

N

K2CO3, DMAc, rt 2 days

N H

2. 1N HCl, Dioxane rt, 4h

O

O

OCH3

N

N

N

N

OMe

1

1

N H

1

N H

1

N H

7

8

1

N H

O

9

H N

10

1

N

H N

11

S

H N

1

N H

H N

1

15

1

16

17

1 1

HN

N H

1

N H

1

N

O

1

22

23

1

N

27

N

OMe

1

N

26

N

N

N 21

OMe

1

25

1

N

N

H N

20

N

N 24

N

19

N

N

H N

N

18

N

N

1

14 1

1

O

H N

1

13

O

O

O

12

HO 1

1

6 - 30

H

O

N H

OMe

OMe

MeO 6

1

1

1

N

N

STAB/DCM

O HN

N

N

R1R2NH

N

OMe

28

N

29

30

Scheme 3. Synthesis of 2-(6-methoxy-9H-purin-9-yl)propan-1-amine analogs (31–55) OCH3

1. Br

OMe N N

OMe

OCH3

N


N H


2

OMe N H

1

31

N H

OMe

1

H N

1

32

O

H N

O

33

1

1

34

1

N H

1

40

1

1

1

HN

42 1

N N

N

N

N H

41

1

N

N N

O

O

N H

1

1

H N

1

1

1

45

49

50

51

39 1

N

O

N

46

1

47

OMe

N

N

N N

O

OMe N

1

N

N

1

HN O

N

OCH3 48

H N 38

N

N

N

O 37

44

1

H N

36

43 1

N

H N

35

HO O

H N

1

31 - 55

O

N

H N

N

N

STAB/DCM

H

1

N

N

R1R2NH

N

N

OMe

OMe

N

N

1

N

N

OMe 52


53

54

55


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Scheme 4. Synthesis of 2-(6-thiomethyl-9H-purin-9-yl)propan-1-amine analogs (56–88) OCH3

1. Br

N

OCH3 K2CO3, DMAc, rt 2 days

N H


SCH3 N N

SCH3

SCH3

N

N

N

N 3

1

N H

56

57 N 1

N H

1

N H

65

N H

58

O 1

1

O

N H

OMe

O

N H

73

74

N H

1

N H

82

N

N

H N

1

75

76

1

N 83

1

69 1

1

63

1

N H 64

84

71 1

N N

79 O

1

85

N H 72

1

N

N N

N

N N

78

OMe

1

N H

O 77

N

N

1

N

N

NH

N H 70

N

1

N

1

N H

62

HN 68

N 1

61

O 1

1

N H

1

1

67

OH

S

O 1

N H

60

HN

N 1

1

N

N

N

N H

66

N H

O N H

59

O 1

1

1

56 - 88

O 1

N H

N

N

STAB/DCM

H

1

N

N

R1R2NH

N


81

OMe

1

N OMe

86

OCH3

80

87

1

N

N 88

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Scheme 5. Synthesis of benzyl (1-(3-aminopropyl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate analogs (89–122) O O


N

NH

O

N

1

N H

1

OMe

1

N H

89

1

H N

H N

90

1

O 98

91

1

99

100

HN

1

107 1

1

115

N

N

1

N H

1

N

117

O

N

N 118

N H

1

N

N

1

N

N 114 N

OMe 1

OMe 121

N

N OCH3

1


N

113

N 120

N H 106

N

OMe

HO 1

105

112

119

N

1

O 111 1

N

97

104

N

110

1

N

Ph

103

H N

96

1

N H

O

N 116

1

N

109 F

N

95

1

O

1

N

N H

HN 94

N H

N H

108

N N

1

1

HN

1

102 1

N

N

H N

1

101

N

H N

1

N 1

O 1

O

1

1

H

O

H N

N

89 - 122

93

HN

HN

N

S 92

1

H N

H N

1

O

STAB/DCM

4

OMe

NH N

R1R2NH

O MeO

O

N


N H

O

O

OCH3

NH

O

O

OCH3

1. Br

N

122


Page 14 of 27

Scheme 6. Synthesis of N-(3,4-dimethylphenyl)-1H-1,2,4-triazole-3-carboxamido-propan-1amine analogs (123–147)

OCH3

1. Br

OCH3


NH N

O

N

O

123 - 147 O

H

OMe 1

1

N H

1

H N

O 132

1

N H

123

1

OMe

1

H N

1

124

OMe

O

H N

O

125

H N

1

126

1

N

N H

133

1

1

N H

134

1

N H

135

N

1

141

1

O

N

1

137

OMe

142

1

1

OMe

143

1

131

1

139 1

N N

N

N

NH

138 1

H N

130 N

N H

N

N

1

O

OH MeO

N H

136

H N

129

OMe

1

N

H N

1

128

MeO

N

H N

O

127

OH

1

H N

1

O

H N

N N

STAB/DCM

5

OMe

N

O

R1R2NH

N N


N NH

NH

NH

N

N H

140

N N

N N

144

145

146

147

Scheme 7. Synthesis of 4-amino-1-(3-aminopropyl)pyrimidin-2(1H)-one analogs (148–157) O O

NH2

NH

O

N

40% NaOH

N

O

MeOH, rt

N

N 1

99, 104, 109, 110, 113 114, 118, 119, 121, 122

NR1R2

148 - 157

OMe

1

1

N H 148

1

149 1

1

N N 154

1

N

N H

1

N 1

N H

150 1

N N O 155

N

OMe

O

Ph

N

151 1

N

N

152

153

N

N

N

N

N

N 156


157

OCH3

O

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Figure 4. Structures of diamine analogs (158–162) OMe

OMe N

N

N

N

N

OMe

N

OMe MeO

O

OMe

N

N

N OMe

N

N

N N H

N

N

160 N

OMe

O

N N

161

N

O N H

N

O

HN

H N

N

O O

N N

N N

162



159

N

NH

O

N

N

O

OMe

N N

N

N

SCH3 N

N

N

158

N

N N

N

N

NH

BIOLOGICAL EVALUATION The reported analogues were tested for in vitro inhibition of the growth of Mycobacterium

tuberculosis H37Rv (Mtb).13 None of the compounds showed appreciable activity at compound concentrations lower than 100 μM. Additionally, 117 available compounds from the set were screened in vitro against three human tumor cell lines (HT29 colon, PC3 prostate, and MDA-MB231 breast).5b Thirty-seven compounds showed toxicity at less than 50 μM in at least one of these cells and examples are given in Table 1. All analogs were submitted (20 mg) to the MLSMR to be screened against a wide range of biological assays (see www.ncbi.nlm.nih.gov/pcsubstance search term Robert Reynolds or PubChem CID numbers provided in Supplementary Materials).



Numerous (i.e. 144 out of 157) analogs included in this publication were tested in a total of 386 bioassays and 74 compounds have shown activity in at least one bioassay. Examples of these activity results are presented in Table-S1 in the Supplementary Materials. Table-2. Effect of Analogs on Cancer Cell Growth Analog 8 17 18 24 26 50 69 70 73 82 83 84 89 90 93 97 100 103 104 109 113 117 118 120 126 127 128 130 131 133 135 141 143 144 145 146

Cancer Cell Line Screen (CC50)a HT29 18.55 ± 0.89 3.47 ± 0.17 35.59 ± 1.34 3.38 ± 0.25 2.34 ± 0.17 24.63 ± 1.42 10.15 ± 1.44 44.30 ± 1.69 20.47 ± 1.91 36.13 ± 2.84 8.63 ± 1.19 8.65 ± 0.40 46.63 ± 4.42 28.85 ± 2.17 29.88 ± 1.30 23.61 ± 2.49 5.81 ± 0.16 18.20 ± 0.48 4.47 ± 0.45 9.48 ± 1.19 24.07 ± 2.41 3.64 ± 0.21 2.74 ± 0.34 47.06 ± 2.47 16.74 ± 2.08 11.94 ± 2.42 32.40 ± 11.14 4.85 ± 0.71 3.66 ± 0.34 6.50 ± 1.34 5.98 ± 0.84 4.51 ± 0.53 13.22 ± 2.06 24.05 ± 6.64 1.87 ± 0.15 47.92 ± 23.05

PC3 >50 8.38 ± 0.38 >50 12.76 ± 0.93 6.79 ± 0.58 >50 >50 >50 >50 >50 27.89 ± 1.49 26.52 ± 5.09 >50 27.71 ± 1.57 >50 >50 13.16 ± 0.53 >50 9.51 ± 0.83 28.58 ± 1.46 47.14 ± 3.70 9.25 ± 1.03 7.94 ± 0.77 >50 >50 44.51 ± 4.50 >50 13.09 ± 0.55 10.83 ± 0.44 29.73 ± 2.11 19.56 ± 0.63 13.58 ± 1.07 >50 >50 6.8 ± 0.40 >50

MDA-MB-231 46.50 ± 2.51 5.76 ± 0.71 >50 7.30 ± 0.86 5.90 ± 0.16 >50 32.60 ± 5.00 >50 >50 >50 26.0 ± 1.89 19.7 ± 0.99 >50 44.68 ± 4.65 >50 >50 15.08 ± 0.84 >50 8.36 ± 0.28 16.17 ± 0.63 >50 17.17 ± 2.39 9.74 ± 0.77 >50 39.01 ± 3.79 28.16 ± 2.17 >50 11.43 ± 43.57 7.98 ± 2.38 16.05 ± 0.78 14.28 ± 1.25 9.99 ± 0.75 >50 >50 5.94 ± 0.52 >50


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aCC 50

= Concentrations in µM of analog required for 50% growth inhibition of cancer cells.

 CONCLUSIONS Herein, we report the synthesis and preliminary biological evaluation of a diverse library of 157 purine, pyrimidine and 1,2,4-triazole amine analogs which were prepared in a high throughput solution-phase parallel reaction format under the Pilot Scale Library Program of the NIH Roadmap initiative. These reactions were performed in 24-well reaction blocks with automatic reagent dispensing under inert atmosphere. All compounds were characterized by 1H NMR and HRMS (ESI), and were checked for purity by HPLC before submission to The Molecular Libraries Small Molecule Repository (MLSMR) at the NIH. Preliminary screening was performed in vitro in our labs against M. tuberculosis H37Rv strain and against three cancer cell lines from colon, breast and prostate that are standard primary screening lines for initial anticancer activity. Antitubercular screens produced no actives below 100 µM. The screening against the cancer cell lines, however, resulted in numerous hits in the three cell lines with CC50 ranges from about 2.0 to 50 µM, sixteen of which gave reasonable phenotypic growth inhibition at

Synthesis of Aza-acyclic Nucleoside Libraries of Purine, Pyrimidine

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