Parallel Solution Phase Synthesis and Preliminary Biological Activity

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Parallel Solution Phase Synthesis and Preliminary Biological Activity of a 5’-Substituted Cytidine Analog Library Omar MOUKHA-CHAFIQ, Robert C. Reynolds, Jacob C. Wilson, and Timothy S. Snowden ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.9b00072 • Publication Date (Web): 31 Jul 2019 Downloaded from pubs.acs.org on August 7, 2019

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ACS Combinatorial Science

Parallel Solution Phase Synthesis and Preliminary Biological Activity of a 5’Substituted Cytidine Analog Library Omar Moukha-Chafiq1*, Robert C. Reynolds2*, Jacob C. Wilson3, and Timothy S. Snowden3 1Chemistry Department, Drug Discovery Division, Southern Research, 2000 Ninth Avenue South, Birmingham, Alabama 35205, United States. 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 3Department of Chemistry and Biochemistry, The University of Alabama, 250 Hackberry Lane, Tuscaloosa, AL 35487-0336, USA

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ABSTRACT: A 109-membered library of 5’-substituted cytidine analogs was synthesized, via funding through the NIH Roadmap Initiative and the Pilot Scale Library (PSL) Program. Reaction core compounds contained -NH2 (2) and -COOH (44 and 93) groups that were coupled to a diversity of reactants in a parallel, solution phase format to produce the target library. The assorted reactants included -NH2, -CHO, -SO2Cl, and –COOH functional groups, and condensation with the intermediate core materials 2 and 44 followed by acidic hydrolysis, produced 3-91 in good yields and high purity. Linkage of the amino terminus of D-phenylalanine methyl ester to the free 5’-COOH of 44 and NaOH treatment led to core library –COOH precursor 93. In a libraries from libraries approach, compound 93 served as the vital building block for our unique library of dipeptidyl cytidine analogs 94-114 through amide coupling of the –COOH group with numerous commercial amines followed by acidic deprotection. Initial screening of the complete final library through the MLPCN program revealed a modest number of hits over diverse biological processes. These hits might be considered as starting points for hit-to-lead optimization and development studies.

NH2

NH2 N H 2N

O O

N

O

2 steps

N

H N

R

O HO

O

OH

NH2

NH2

NH2

N

N

N

O N O OH OH 45-91

O

HO

2 steps

O N O O 44

O

3-43

2

R

N

O

HO C O 2 steps 2 Ph

H N

O N O O

O

93

NH2 O O 2 steps R Ph

H N

N O N O

O

HO OH 94-114

KEYWORDS: cytidine nucleoside analogs, solution phase, diversity libraries, biological activities.


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INTRODUCTION Nucleosides are fundamental biological components that interact with a significant portion of the human proteome, including peptide ligases, tRNA synthetases, polymerases, kinases, reductases, motor proteins, membrane receptors, and structural proteins.

With advances in both high

throughput chemical synthesis and screening, these key building blocks are increasingly relevant today, and they are finding utility in the discipline of chemical biology as well as modern drug discovery. As privileged and biologically relevant scaffolds, nucleosides continue to be useful as probes for novel and essential nucleoside-dependent processes. In fact, over the past few decades, new nucleoside-based agents have been discovered that are active for viral infections, cancers, cardiovascular disorders, pain, etc. A number of drugs have resulted from targeting nucleoside metabolism, including potent antiviral and anticancer drugs.1-3 As with many drug classes, however, the increasing resistance of pathogens and cancers to current nucleoside-based drugs, the severe side effects sometimes produced by nucleoside antimetabolites, and the plethora of new nucleoside-dependent targets resulting from genetic sequencing highlight the continued need for new and unique nucleoside diversity sets relevant to this fundamental slice of the proteome. Such diverse nucleoside-based libraries will be potentially useful not only as probes for new chemical biology approaches to assess fundamental pathways and the role of target proteins in metabolism, growth, and drug resistance, but may also serve as a reservoir for new drug leads. Nucleosides are relatively low molecular weight compounds that can serve as superb starting components for diversity-oriented synthesis to prepare interesting and highly diversified small molecules with various groups having unique and varied spatial orientation for probing biological diversity space. In spite of their rich history and utility, however, nucleoside analogs are not commonly present in research or commercial chemical libraries.


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Advancements in high-throughput chemistry have rarely been applied to nucleoside synthesis but may allow rapid preparation of diverse and biologically relevant libraries featuring this privileged scaffold. Thus, the goal of our PSL grant was to prepare novel and diverse nucleosides not only for our own use, but also as a public resource for access via government-sponsored chemical libraries and screening programs. It is notable that standard nucleosides can be promiscuous toxins via phosphorylation of the 5’-hydroxyl group and entry into numerous crucial metabolic pathways. As such, the perception that nucleoside drugs are generally nonselective and potently toxic through cross reactivity through multifarious DNA and RNA pathways has led to the stigma associated with this class. In sharp contrast, however, there are abundant cases of relatively discriminating nucleosides, both simple and complex, that exhibit specific biological activities.5,6 The naturally occurring nucleoside antibiotics, are one such example. As a class, they show widely varying and compelling activities against numerous specific targets such as the protein synthesis machinery, glycosyltransferases, and methyltransferases as well as many other critical protein targets.4-7 Of particular interest are the 5’-substituted peptidyl nucleoside analogs containing a diversity of amino acids and amino sugars, leading to the vigorous research to isolate and prepare new amino acid and perptide-substituted nucleosides.7-11 Herein, we report the preparation of a focused set of cytidine compounds inspired by the broad class of natural peptidyl nucleosides6,12,13 including capuramycin, the polyoxins (tunicamycin and nikkomycin), the muramycins and the mureidomycins. EXPERIMENTAL DESIGN AND DISCUSSION We recently described the preparation and testing of a variety of adenosine and uridine analogs that demonstrated diverse biological activities in early MLPCN assays.14 We now report the


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synthesis of a cytidine analog library derived from intermediates 2 (-NH2 reactant) and 44 and 93 (-COOH reactants) (Schemes 1 & 2) using solution phase, parallel chemistry. Our goal was to target variations around the 5’-position to mimic structures represented by the natural nucleoside antibiotics that demonstrate a variety of interesting activities. This task was accomplished through robust coupling chemistry either utilizing 5’-aminocytidine (2) or the 4’COOH analog of the nucleoside to produce amines (via reductive amination), amides (peptide coupling), or sulfonamides (via diverse commercial R-SO2Cl reagents). In a libraries-fromlibraries approach, D-phenylalanine was randomly chosen to be coupled to 44 to give the chiral amino acid intermediate. Further coupling through the free D-Phe carboxylic acid yielded a diverse array of amide-linked analogs potentially relevant to the natural nucleoside antibiotics, since the more complex natural products oftentimes contain chiral linkages (amino acids, carbohydrates, etc.) that are considered critical for biological uptake and targeting. It was essential to demonstrate that our new library was unique in order to obtain PSL funding. Hence, we first selected a commercial set of -CHO, -NH2, -COOH, and –SO2Cl containing reactants that were obtainable in satisfactory amounts and at an acceptable cost to drive downstream synthesis and diversity. This set was further filtered by eye for diversity and desired reactivity to work in the designed coupling reactions and provide the expected diversity of nucleoside products. Finally, the 3D representations were further assessed using Tanimoto structural similarity relative to the current MLSCN library set at that time. Tanimoto similarity is one measure to determine similarity between compounds and compound sets using compound fingerprints. The score ranges from 0 to 1 with 0 (no similarity) and 1 (100% similarity). A Tanimoto value of 0.7 to 1.0 suggests that two molecules are highly similar to identical. Every proposed structure was compared with the total MLSCN library (as of the year 2010 - 197,873 discrete molecules). Only 9.3% of our total


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proposed set had a Tanimoto value greater than 0.7, indicating that the new materials were significantly divergent from the MLSCN library available in 2010. Furthermore, 36% of the new nucleosides had Tanimoto values below 0.5, indicating significant diversity relative to the current MLSCN library. 5’-Amino and 5’-carboxylic nucleosides are common staring points to prepare diverse nucleosides for screening.12-20 Consequently, the nucleoside 2 and nucleosides 44 and 93 were chosen as three diversification reagents for robust reductive aminations, sulfonation reactions or amidations in order to synthesize our unique cytidine-based library as depicted in Schemes 1-3.

Scheme 1: Synthesis of Analogs 3-43 NH2

NH2

NH2

NH2

N

N

N

N

O

N

HO

i)

O O

N

TsO

O

ii)

O

O

O

1

N

N3 O O

O

O

iii)

O

O O

O

1b (82%)

1a (72%)

N

H 2N

O

2 (74%) iv) and v)

NH2 N H N

R

N O HO

OH

35-43 (65-78%)

N

N O

R

S

H N

N

O

R

H N

N

HO

O

O

O

O O

O

NH2

NH2

OH

30-34 (71-75%)

HO

OH

3-29 (67-85%)

Reagents and conditions: i) TsCl, pyridine; ii): NaN3, DMF, 50 oC. iii): NH4OOCH, Pd-C 10%, MeOH. iv) aldehyde derivative MeOH, 0-40 oC, NaBH4; sulfonyl chloride derivative, DMF, Cs2CO3 or carboxylic acid derivative, HATU, DIEA, CH3CN; v) 50% HCO2H, 70 oC.


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NH2

NH2

N

N

N

N

HO

O

O

HO

i)

O

O

N

O

O

O

O

HO

O

O

OH

45-91 (66-82%)

44 (86%)

1

N O O

R

ii), iii)

Reagents and conditions: i) cat. TEMPO, BAIB, NaHCO3, MeCN:H2O, r.t.; ii): R = amine derivative, HATU, DIEA, CH3CN.; iii) 50% HCO2H, 70 oC.


NH2 N HO

O N O O 44

O

O

MeO2C i)

Ph

H N

N O N O O

O

92 (88%)

NH2

NH2 O

HO2C ii)

Ph

H N

N O N O O

O O

O

93 (75%)

iii)

R Ph

H N

N O N O HO

O

OH

94-114 (65-73%)

Reagents and conditions: i): Methyl (R)-2-amino-3-phenylpropanoate, HATU, DIEA, MeCN. r.t.; ii): 1N NaOH, dioxane, r.t.; iii): a: R = amine derivatives, HATU, DIEA, MeCN, r.t.; b: 50% HCO2H, 70 oC, 2 h.

5’-Amino-5’deoxycytidine (2) was prepared from 1 in three steps via a reported procedure and was identical in all respects to the reported material.20 Reductive amination is efficient and easily adaptable to parallel chemistry, and we utilized this approach to couple pure compound 2 with a variety of diverse, commercially available -CHO containing reagents. Coupling was accomplished in MeOH over molecular sieves to drive intermediate imine formation through water removal; the drying reagent was critical for higher yields. A Radleys 12-place carousel reaction station was used for parallel production of the libraries. Chemistry was carried out at room temperature unless indicated as certain reactions with poorly soluble aldehydes required warming to 40 oC for 10 minutes. The aldimine intermediates were treated in situ by careful addition of solid NaBH4. After 30 minutes, the reaction was adsorbed onto silica gel without further workup and dried followed


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by automated flash chromatography purification. Acid catalyzed removal of the acetonide protecting group in 50% formic acid yielded the final compounds 3-29 (Scheme 1) in 67-85% yields. Target compounds are presented in Charts 1-3.


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Chart 1: Structures of Analogs 3-43 NH2 N H N

N

R HO

Compound 3

N

N

O

OH

Compound

R

17

N

O

Compound

R S

N

O

R S

Me

31

N

S

18

4

O

O

32

Me

S O O O O S

NC Ac

5

19

MeHNO2S Cl

Cl

6

20

O

7

OiPr

OMe

Cl

O O S

N

33

O

OMe

O

34

O

S

O

CN

CN

O

21

O O S

35

NH2

F 3C

8

9

O

22

HO

Br

BnO

23

O Cl

NH2

S

NN

Me

N

36

CH2

O

N

37

N

NHCHO

OBn

10

11

12

13

Me

OMe

O

Cl O

N N

25

MeN

26

Me2N

N

38

O

N

O

39

O N H H O

Me

OMe

N O S

N

24

O

NH2

S

40

O

N H Me

MeO

27

Me

ON

41

MeHN

HO

14 S

28

N

HO

42

O

H2 C

O

O

O

O O O

15

MeO F

29

HC

16

43

O

N

O Cl

S

NH2

NHCHO

O Me

30

Me

S

S O


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Chart 2: Structures of Analogs 45-91 NH2 N N O O

R

HO

Compound 45

ON H N

46

PhO

47

N

OH

Compound

R MeO

O

N

NH

Compound

R NN

61

H N

N Pr

H N

N

O

F

N

N

49 Me

OMe N N

Me

NH

N

64 65

NH Me N N

Me

N

MeO

N

O

52

63

NH

N

53 O

55

N N

F

H N

66 67 68

N

69

O H N

N Me H

82

S

N

Cl

N

H N

N F 3C MeO MeO

Me

83

Me N

84

N

H2NOC

85

OH

Me

NH

N

F 3C

OH N H

86

N

N

Me2N

N

87

N

88

OCHO NH

N

N

HO

HN

59 60

H N

Me O

Me O

N H

O Me N N

73

76

N

N

89

OHCO

HN

Me

Me N

74 75

H N

O

N

Me Me

O

58

OCHO

Me Me

NH

72 NH

F

OH

Me

O

57

N

N

CF3

O

71

N H

N

O

70

F 3C

56

Me N

N

80 81

F 3C

Me

N H

Me

79

NH

MeO MeO

MeO O

54

N S

Me N

48

51

N

O

N H N H O

78

O NH

N

50

77

O

62

R

O N

HO

N

F F

N

N

N

90 91

HO MeS

N H OH NH

Me


10

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Chart 3: Structures of Analogs 94-114 NH2 N

H N

O R Ph

O N O

HO

O

OH

R

Compound

R

94

OH

105

H2NOC

N H

106

S N

107

F F

108

N

95 96 97 98

F 3C

Compound

Me H Me N

N O H

OMe OMe OMe

N H

HN

N

100 101

O O

102

S N

103

MeO

104

MeO

O

N N

109 N H

110

N H

111

N H

OMe Me N

N

N N

O N

N

Me2N O N N

112

N H

N

O N F 3C

O

Me O

N

MeO O

N O H

O

99

N

113

O

114

HO

O N N

N

N

N

N

OMe


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A 24-position MiniBlock XT vessel was employed to prepare our library members 30-43 (Chart 1), 45-91 (Chart 2) and 94-114 (Chart 3) in solution phase. A Tecan liquid handling system was used for dispensation, retraction, and aspiration with a compatible 17 × 110 mm test tube carousel and a Genevac evaporation system. In this format, five sulfonyl chlorides were reacted with 2 to prepare the sulfonylamide cytidine analogs 30-34 (Scheme 1) using the solvent DMF and CsCl (base) for coupling. The isopropylidene blocking groups were removed as described above. HATU (1 equiv) was used for amide coupling of 2 with nine -COOH compounds catalyzed by DIEA (1.5 equiv) in acetonitrile (three hours) to prepare amide-linked compounds 35-43 in 6578% yields (Scheme 1) after acid mediated deprotection.. These conditions with intermediate 44 gave amides 45-91 in 66-82% yields (Scheme 2). Compound 44 was prepared by reported methods using catalytic amounts of TEMPO and BAI in acetonitrile-water (1:1).12a Compound 44 was coupled to the free amino group of D-phenylalanine-COOMe 92 (Scheme 3) in 88% yield. Hydrolysis with 1N NaOH in dioxane at 25 oC gave the -COOH intermediate 93 in 75% yield. Deprotection with 50% formic acid at 70 oC afforded a high yield of nucleoside 94. Target 93 was designed from a “libraries from libraries” standpoint, and produced a site of further diversity expansion through another amide coupling with diverse amines to produce targets 95114 (Scheme 2). Coupling of 93 with 20 diverse amines and HATU/DIEA produced the desired targets in 65-73% yield and high purity after acid catalyzed removal of the isopropylidene protecting group and purification. Analysis of all products involved 1H NMR, HPLC, and mass analysis. Purity ranged from 90100%, and the average purity was 97%. Comparison of the NMR spectra of crude 93 as its carboxylate salt with spectra of the corresponding crude epi-93 carboxylate salt -prepared by amide coupling of 44 with methyl (S)-2-amino-3-phenylpropanate to form epi-92, purification, then


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saponification using 1N LiOH in dioxane - established that the hydrolysis of 92 to 93 proceeds without epimerization.22 BIOLOGICAL EVALUATION All biological screening was performed via government grants and contracts to external and independent academic laboratories through the MLPCN program, and all results can be readily accessed by a specific assay name in PubChem Assay or through the compound database via www.ncbi.nlm.nih.gov/pcsubstance (search term Robert Reynolds). Certain nucleosides (see Table 1) afforded a variety of modest activities in the primary screens. Table 1: Selected Results from PubChem Bioactivity Analysis Compound 54, 59, 65, 97, 101, 103, 107 and 112 28 35 39 42

54

59 and 85 65

Assay Title in PubChem “High-Throughput-Screening (HTS) to identify inhibitors against Sialic Acid Acetyl Esterase (SIAE)” “Discovery of small molecule inhibitors of the oncogenic and cytokinetic protein MgcRacGAP.” “HTS to identify agonists of the mouse 5hydroxytryptamine (serotonin) receptor 2A (HTR2A)” “HTS to identify inhibitors of phospholipase C isozymes (PLCgamma1).” “Small Molecule Inhibitors of FGF22Mediated Excitatory Synaptogenesis & Epilepsy Measured in Biochemical System Using RT-PCR.” “HTS to identify positive allosteric modulators (PAMs) of the human cholinergic receptor, muscarinic 4 (CHRM4).” “THS to identify modulators of interaction between CendR and NRP-1.” “Counterscreen for inhibitors of 5meCpG-binding domain protein 2 (MBD2).”

Biological results 26%, 52%, 26% 45%, 17%, 47%, 45% and 23%, respectively at 9.6 µM 34% inhibition at 40 µM 29% inhibition at 7.6 µM 10% inhibition at 12.2 µM 43% inhibition at 9.9 µM

20% activation at 3 µM 40% inhibition at 25 µM 43% inhibition at 4.4 µM


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57 and 71 80 90

102

“DENV2 CPE-Based HTS Measured in Cell-Based and Microorganism Combination System” “HTS to identify D3 Dopamine Receptor Antagonist.” “HTS to identify inhibitors of the interaction of nucleotide-binding oligomerization domain containing 2 (NOD2) and the receptor-interacting serine-threonine kinase 2 (RIPK2).” “HTS to identify inhibitors of protein arginine methyltransferase 1 (PRMT1).”

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70% and 87 inhibition, respectively, at 9.99 µM 40% inhibition at 2.3 µM

28% inhibition at 5 µM 21% inhibition at 2.8 µM

For example: 5’-carboxamide nucleoside analogs 54, 59 and 65, and di-peptidyl compounds 97, 103, 107 and 112 showed modest, lead-like inhibitory activity against Sialic Acid Acetyl Esterase (SIAE) (Table 1). This enzyme could serve as a target for the pharmacological induction of immune activation to empower B cells that are specific for weak epitopes and to enhance T cell memory responses. It has been shown that excitatory and inhibitory synapses can be organized in the brain by two fibroblast growth factor (FGF) family members, FGF22 (excitatory) and FGF7 (inhibitory). . Remarkably, FGF22-deficient mice are resistant to epileptic seizures, and FGF7-deficient mice are prone to them.22 These results indicate that the identification of small molecules that inhibit FGF22-mediated excitatory synapse formation or those that can activate FGF7-mediated inhibitory synapse formation may lead to new treatment approaches for epilepsy. Analog 42 showed modest inhibition (43%) against FGF22 at 9.9 µM (Table 1) and may be used as a lead for developing potent FGF inhibitors due to its unique structure. In addition, protein arginine methyltransferase 1 (PRMT1) activity has been linked with a number of human health conditions including cardiovascular disease, cancer, infectious disorders and autoimmune conditions.23 Di-peptidyl analog 102 showed modest activity against PRMT1 with


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21% inhibition at 2.8 µM (Table 1) and may warrant further investigation as this structure is unique from other reported inhibitors.23 CONCLUSION Preparation and initial screening of a unique library of 109 cytidine-based compounds is reported. Application of newer higher throughput equipment and technologies were utilized in the preparation of this small-sized diversity set. Targets were sent to the MLPCN for screening, and early assay results showed modest activities against the targets SIAE, FGF, and PRMT1. The PRMT1 hit, while only modestly active, is unique relative to reported PRMT1 inhibitors, and we are currently preparing more material as well as analogs for confirmation of activity in advanced assays. All assay data and protocols can be viewed by visiting PubChem Substance. ASSOCIATED CONTENT Supporting Information detailing the general experimental procedures and analytical data for the libraries is available free of charge on the ACS Publications website at DOI:. General Experimental Section

Page S3

General Procedures & Analytical Data for Compounds 3-29

Pages S3-S8

General Procedures & Analytical Data for Compounds 30-34 & 35-43

Pages S8-S11

Procedure & Analytical Data for Compound 44

Page S11

General Procedures & Analytical Data for Compounds 45-91

Pages S11-S19

General Procedure & Analytical Data for Compounds 92, epi-92, and 93 Pages S19-S20 General Procedures & Analytical Data for Compounds 94-114

Pages S20-S24

References

Page S24

NMR Spectra of Representative Products Pages

S25-S51

Comparative NMR Epimerization Study Pages

S52-S54


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AUTHOR INFORMATION Corresponding Author *Omar Moukha-Chafiq: [email protected] *Robert C. Reynolds: [email protected] ORCID Omar Moukha-Chafiq: 0000-0002-4906-1643 Robert C. Reynolds: 0000-0001-9413-2247 Jacob C. Wilson: 0000-0001-9777-9898 Timothy S. Snowden: 0000-0002-6838-1220 Notes The authors have no financial conflicts. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. All authors contributed equally. Funding Sources This investigation was supported by NIH grant No. 1P41 GM086163-01 (entitled: Pilot-Scale Libraries Based on Nucleoside Templates for the ML Initiative, Robert C. Reynolds, P.I.). ACKNOWLEDGMENT


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We appreciate the input of James M. Riordan, Jackie Truss, Mark Richardson and David Poon of the Molecular and Spectroscopy Section of Southern Research Institute for analytical support. The reported work was sustained by NIH grant No. 1P41 GM086163-01 (entitled: Pilot-Scale Libraries Based on Nucleoside Templates for the ML Initiative, Robert C. Reynolds, P.I.). Dr. Robert C. Reynolds would like to thank Drs. Boris Pasche and Ravi Bhatia for support of this research project via start-up funds provided through the Division of Hematology and Oncology at the University of Alabama at Birmingham.

ABBREVIATIONS PSL, Pilot Scale Library; NIH, National Institutes of Health USA; tRNA, transfer ribonucleic acid; MLPCN, Molecular Libraries Probe Production Centers Network; DNA , deoxyribonucleic acid; MLSCN, Molecular Libraries Screening Centers Network; DMF, N,N-dimethylformamide; HATU, [(2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate]; DIEA, N,N-diisoproplyethylamine; TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxyl; BAIB, [bis(acetoxy)iodo]benzene; NMR, nuclear magnetic resonance; HPLC, high performance liquid chromatography; FGF, fibroblast growth factor; PRMT1, Protein arginine methyltransferase 1; SIAE, Sialic Acid Acetyl Esterase; TLC, thin layer chromatography; TMS, tetramethylsilane. REFERENCES 1. (a) Perigaud, C.; Gosselin, G.; Imbach, J.-L. Nucleoside Analogues as Chemotherapeutic Agents. Nucleosides Nucleotides 1992, 11, 903-945. (b) Jacobson, K. A.; van Galen, P. J. M.; Williams, M. Adenosine receptors: pharmacology, structure-activity relationships, and therapeutic potential. J. Med. Chem. 1992, 35, 407-422. (c) Williams, M.; Jarvis, M. F.


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of

fibroblast

growth

factors

in

epileptogenesis-associated

circuit

rearrangements. Front Cell Neurosci. 2013; 7: 152. 22. See Supporting Information for NMR spectra of the crude ester hydrolysis products 93 and epi-93.


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TOC

NH2

NH2 N H 2N

O O

N

O

2 steps

N

H N

R

O HO

O

OH

NH2

NH2

NH2

N

N

N

O N O OH OH 45-91

O

HO

2 steps

O N O O 44

O

3-43

2

R

N

O

HO C O 2 steps 2 Ph

H N

O N O O

O

93

NH2 O O 2 steps R Ph

H N

N O N O

O

HO OH 94-114


21

Parallel Solution Phase Synthesis and Preliminary Biological Activity

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