Exploring Human Parainfluenza Virus Type-1 Hemagglutinin

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Exploring human parainfluenza virus type-1 hemagglutininneuraminidase as a target for inhibitor discovery. Ibrahim Mustafa El-Deeb, Patrice Guillon, Moritz Winger, Tanguy Eveno, Thomas Haselhorst, Jeffrey C. Dyason, and Mark von Itzstein J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500759v • Publication Date (Web): 24 Aug 2014 Downloaded from http://pubs.acs.org on September 1, 2014

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Journal of Medicinal Chemistry

Exploring human parainfluenza virus type-1 hemagglutinin-neuraminidase as a target for inhibitor discovery. Ibrahim M. El-Deeb‡*, Patrice Guillon‡*, Moritz Winger‡*, Tanguy Eveno, Thomas Haselhorst, Jeffrey C. Dyason and Mark von Itzstein* Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, 4222, Australia KEYWORDS Parainfluenza, Hemagglutinin-Neuraminidase, Neu5Ac2en, Sialic acid, Neuraminic acid, Inhibitor.

ABSTRACT. Human parainfluenza virus type 1 is the major cause of croup in infants and young children. There is currently neither vaccine nor clinically effective treatment for parainfluenza virus infection. Hemagglutinin-Neuraminidase glycoprotein is a key protein in viral infection, and its inhibition has been a target for Neu5Ac2en-based inhibitor development. In this study we explore the effect of C-5 modifications on the potency of Neu5Ac2en derivatives that target the human parainfluenza type-1 hemagglutinin-neuraminidase protein. Our study demonstrates that the replacement of the Neu5Ac2en C-5 acetamido moiety with more hydrophobic alkane-based moieties improves the inhibitory potency for both hemagglutinin-neuraminidase functions. These findings shed light on the importance of C-5 substitution on Neu5Ac2en, in the design of novel sialic acid-based inhibitors that target human parainfluenza type-1 hemagglutinin-neuraminidase.

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INTRODUCTION The human parainfluenza viruses (hPIVs), members of the Paramyxoviridae family, are important respiratory

tract

pathogens

that

cause

severe

disease

in

infants,

young

children,

the

immunocompromised and elderly.1-4 Human parainfluenza virus type 1 (hPIV-1) is the major cause of croup in infants and young children.1,5 Other manifestations for hPIV-1 infection include bronchiolitis, pneumonia and tracheobronchitis.6 There is currently neither vaccine nor clinically effective drug for protection against or treatment of infections by these viruses. hPIV infection is initiated by virus adhesion to a target cell through sialoglycoconjugate recognition by the viral receptor-binding glycoprotein hemagglutinin-neuraminidase (HN). Subsequently, the HN protein activates the fusion protein (F) and triggers virus-cell membrane fusion.7,8 Multiple steps then lead to virus protein production and assembly of virion progeny at the infected cell surface. Finally, the virion progeny are released by the action of the HN sialidase activity.9-11 OH

OH H

OH O

COONa

OH H

O

COONa

OH HN

OH AcHN R

1: R = N3 2: R = OH 3: R = NH2 4: R = NH(C=NH)NH2 (zanamivir)

O

R

5: R = N3 (BCX 2798) 6: R = OH 7: R = NH2 8: R = NH(C=NH)NH2

Figure 1. Structures of the benchmark sialidase inhibitors and their C-5 modified analogues. As a result of multiple key roles of HN in the parainfluenza virus replication cycle (binding, fusion activation and virion release), the HN protein has been considered an attractive target for hPIV inhibitor development.12 While several reports12-17 on aspects of the synthesis and evaluation of neuraminic acidbased inhibitors (for example 1-5, Fig. 1) have appeared over the years, none of these inhibitors have progressed to the clinic. The most potent hPIV-1 inhibitor reported to date is BCX 2798 (5)12,13,17 and it 2 ACS Paragon Plus Environment

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was designed using the X-ray structure of the HN protein from the related Newcastle Disease Virus (NDV) in complex with the broad spectrum sialidase inhibitor 2-deoxy-2,3-didehydro-Nacetylneuraminic acid (Neu5Ac2en, 2).13 Although good efforts have been made to demonstrate the efficacy of 5 against hPIV-1 infection, there is a lack of available structure activity relationship (SAR) information. Since the discovery of 5, a number of Neu5Ac2en analogues with variable substituents at C-4 have been synthesized and screened against hPIV-1.14-16 None of these inhibitors displayed higher potency than 5, however, the variations in the substituents’ size, electronic and hydrogen bonding effects have led to a better understanding of the nature of the cavity oriented towards the C-4 position of Neu5Ac2en. In contrast, and in spite of the various reports describing the synthesis of C-5 modified Neu2en derivatives,18-20 the effect of C-5 substituents in Neu2en derivatives on hPIV-1 HN activity remains relatively unexplored. In this multidisciplinary study we use computational chemistry to better understand the engagement of the C-4/C-5 functionalities within the hPIV-1 HN active site to advance SAR information. Furthermore, we explore substitutions that influence inhibitor potency by (a) combining the C-5 isobutyramido modification of 5 with different C-4 substituents and (b) replacing the C-5 isobutyramido moiety with alternative alkylamides. Thus, the incorporation of a C-5 isobutyramido moiety to the well-known sialidase inhibitors 2, 3 and 4 was undertaken. Consequently, the three well-known sialidase inhibitors 2-4, 4-azido-4-deoxy-Neu5Ac2en (1), and their corresponding C5-modified analogues 5-8, were synthesized and screened against hPIV-1 HN. Finally, we report the synthesis and biological evaluation of novel C-5 modified BCX 2798 (5) analogues to evaluate the effect of C-5 isobutyramido group replacement with other alkylamides on HN inhibitory potency.



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RESULTS AND DISCUSSION Computational Chemistry. The influence of the C-5 alkylamide on the binding efficiency of Nacetylneuraminic acid-based derivatives to the hPIV-1 HN protein was explored by Molecular Dynamics (MD) simulations of inhibitor 3 and its C-5 isobutyramido analogue 7 in complex with a hPIV-1 HN homology model. From inspection of the homology model we identified that both the Neu2en C-4 and C-5 functional group binding domains have significant hydrophobic character. Consequently, incorporating substituents of appropriate size with increased hydrophobic character at both of these positions on the Neu2en framework should lead to improved potency. Table 1. Calculated interaction energies from MD simulations of hPIV-1 HN in complex with 3 and 7.

hPIV-1 HN (3) / kJ mol-1

hPIV-1 HN (7) / kJ mol-1

-506.74 ± 12.80

-546.23 ± 14.71

The average interaction energies of 3 and 7 were calculated from these MD studies (Table 1) and 7 had a lower interaction energy value than that determined for 3. This outcome suggests that there is a predicted enhanced binding efficiency as a direct result of the incorporation of the bulkier hydrophobic isobutyramido moiety. As can be seen in Figure 2 (Panel B), the C-5 isobutyramido group is comfortably oriented within the C5 binding domain of the hPIV-1 HN active site and from further inspection appears to take advantage of additional van der Waal interactions within this region. The results obtained from MD simulations strongly suggest that the contribution of the C-5 substituent to inhibitor potency is significant and will facilitate the design of more potent inhibitors.


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These data taken together provide a strong rationale to explore the synthesis and evaluation of novel C-5 modified compounds as potential inhibitors of hPIV-1 HN.

Figure 2. MD Simulations of the homology model of hPIV-1 HN suggest that the C-5 binding domain within the HN can accommodate bulky alkyl moieties that enhance inhibitor potency. Panel A shows the orientation of the C-5 acetamido moiety in the active site, Panel B shows that a C-5 isobutyramido moiety is well-accommodated within the same domain. Chemistry. Our computational chemistry study inspired us to investigate the notion that the C-5 moiety plays a critical role in hPIV-1 HN inhibitor potency. Thus, we have synthesized and evaluated a range of C-5 and C-4/C-5 Neu5Acyl2en HN inhibitors. The synthesis of inhibitors 5 and 6 was achieved from the known intermediates 9 (Ref 21) and 10 (Ref 22). As shown in Scheme 1, the protection of the NHAc group at C-5 in 9 and 10 by treatment with di-tertbutyl dicarbonate (Boc2O) in the presence of a catalytic amount of DMAP in anhydrous THF provided intermediates 11 (Ref 20) and 12, in 96% and 71% yield, respectively.



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Scheme 1. Synthesis of compounds 5 and 6a OAc OAc H

O

OAc OAc H

COOCH3

a

OAc AcHN R

O

R

11: R = N3 12: R = OAc OAc OAc H

COOCH3

d

O

COOCH3

e

OAc H2N

R

R

13: R = N3 14: R = OAc

15: R = N3 16: R = OAc

OAc OAc H

OH O

COOCH 3

f

OAc HN O

R

17: R = N3 18: R = OAc

a

b, c

Boc

OAc BocHN

COOCH3

OAc Ac N

9: R = N3 10: R = OAc OAc OAc H

O

OH H

O

COONa

OH HN O

R

5: R = N3 6: R = OAc

Reagents and conditions: (a) (Boc)2O, DMAP, THF, 60 °C, o/n, (11, 96%; 12, 71%) (b)

NaOCH3/CH3OH, rt, 3h; (c) Ac2O, pyridine, rt, o/n, (13, 63%; 14, 81% “yields over 2 steps”) (d) TFA, DCM, rt, o/n, (15, 85%; 16, 90%) (e) Isobutyryl chloride, Et3N, DCM, rt, 4 h, (17, 91%; 18, 84%) (f) NaOH, MeOH/H2O (1:1), rt, o/n, (5, 82%; 6, 94%). The deacetylation of these intermediates, by treatment with NaOCH3 and subsequent re-O-acetylation with acetic anhydride in pyridine, yielded the O-acetylated-N-Boc-protected intermediates 13 and 14 in 63% and 81% yield, respectively. Removal of the Boc-protecting group was achieved by treatment of 13 and 14 with TFA in DCM to provide the 5-amino derivatives 15 and 16, in 85% and 90% yield, respectively. Acylation of these amines was readily achieved by treatment with isobutyryl chloride in the presence of triethylamine in DCM and provided 17 and 18 in 91% and 84% yield, respectively. Subsequent deprotection of 17 and 18 by treatment with NaOH gave the reported, but not fully characterised, BCX 2798 (5)23 and 6 in 82% and 94% yield, respectively.


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The synthesis of inhibitor 7 was accessed from intermediate 17 (Scheme 1). Thus, catalytic reduction of the 4-azido group of 17 was achieved by exposure to a H2 atmosphere in the presence of Lindlar catalyst in EtOH to provide 19 in 77% yield (Scheme 2). The deprotection of amine 19, by treatment with NaOH in a mixed solvent of MeOH/H2O, provided the deprotected inhibitor 7 in 61% yield. Scheme 2. Synthesis of compound 7a OAc OAc H

a

17

OH O

COOCH3

b

OAc HN O

OH H

O

OH HN

NH2

O

NH2

7

19

a

COONa

Reagents and conditions: (a) H2/Lindlar catalyst, EtOH, rt, o/n, 77%; (b) NaOH, MeOH/H2O (1:1), rt,

o/n, 61%. Scheme 3. Synthesis of compound 8a OAc OAc H

19

a

OH O

COOCH3

b, c

OAc HN HN O

NBoc

OH H

O

OH HN HN O

NHBoc

20

a

COONa

NH NH 2

8

Reagents and conditions: (a) N,N'-Di-Boc-1H-pyrazole-1-carboxamidine, Et3N, MeOH, argon, rt,

o/n, 73%; (b) TFA, DCM, rt, o/n, 69%; (c) NaOH, MeOH/H2O (1:1), rt, o/n, 61%. The synthesis of compound 8 (Scheme 3) was achieved from the key intermediate 19 (Scheme 2). Thus, 19 was suspended in a methanolic solution of N,N-Di-Boc-1H-pyrazole-1-carboxamidine at rt o/n to provide the 4-(N,N`-di-Boc-guanidino)-4-deoxy-Neu5Ac2en derivative 20 in 73% yield. The deprotection of Boc groups by treatment with TFA in DCM, followed by stirring in a (1:1) mixture of



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methanolic water at pH 13-14 (adjusted by addition of NaOH) afforded the final deprotected 4guanidino product 8. To extend our study on the influence of the nature of the C-5 alkylamide moiety on inhibitor potency, we prepared a number of BCX 2798 analogues with an azido moiety at C-4 and a variety of amides at C-5 (Scheme 4). The synthesis of these novel amides was achieved through reaction of the common key precursor 5-amino-4-azido-4-deoxy-Neu2en intermediate 15 with selected acyl chlorides that introduced a range of hydrophobic and more hydrophilic functionalities (Scheme 4). Scheme 4. Synthesis of 22a-e and 24a OAc OAc H

a

15

O

R O

COOCH 3

OAc HN N3

O

HO

COOCH3

OAc HN

c OAc OAc H

O

N3

21a: R = Cyclopropyl 21b: R = Cyclobutyl 21c: R = CH(CH3)CH2CH3 21d: R = C(CH3)=CHCH3 21e: R = C(OAc)CH3 b

23 OH

b OH

OH H

O

OH HN HO

O

N3

O

COONa

OH HN R

24

a

COONa

OH H

O

N3

22a: R = Cyclopropyl 22b: R = Cyclobutyl 22c: R = CH(CH3)CH2CH3 22d: R = C(CH3)=CHCH3 22e: R = C(OH)CH3

Reagents and conditions: (a) Acyl chloride, Et3N, DCM, rt, 4h, (21a: 82%; 21b: 93%; 21c: 75%; 21d

77%; 21e: 78%); (b) NaOH, MeOH/H2O (1:1), rt, o/n, (22a: 78%; 22b: 89%; 22c: 68%; 22d: 90%; 22e: 88%; 24: 72%); (c) 3-Hydroxy-2-methylpropionic acid, EDCI, HOBT, DMF, rt, o/n, 63%. Thus, acylation of the free amino group in 15 was achieved by treatment with the appropriate acyl chloride in the presence of Et3N in DCM, providing the protected amides 21a-e in good yields. 8 ACS Paragon Plus Environment

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Deprotection of these amides using classic Zemplén conditions afforded the final products 22a-e. The coupling of amine 15 and 3-hydroxy-2-methylpropionic acid in DMF was achieved by treatment of the amine

with

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

(EDCI)

in

the

presence

of

1-

hydroxybenzotriazole (HOBT), affording the protected amide 23 in 63% yield. Subsequent deprotection by treatment with NaOH provided the desired amide 24 in 72% yield. Biological Screening. In order to investigate the effect of replacing the C-5 acetamido group present in the known sialidase inhibitors 2-4 with an isobutyramido group; inhibitors 1-4 and their C-5 isobutyramido analogues 5-8 were evaluated for their capacity to block both the neuraminidase and hemagglutinin functions of the hPIV-1 HN protein, using neuraminidase inhibition (NI)13,24-26 and hemagglutinination inhibition (HI)13 assays, respectively.

Figure 3. The NI (plain) and HI (strips) IC50 values of the inhibitors 1-8. The dotted red line represents the maximum threshold for HI assay (250 µM). Within the C-5 acetamido series of inhibitors, the potent influenza virus sialidase inhibitors 3 and 4 displayed the weakest inhibition for hPIV-1 HN neuraminidase and hemagglutinin activities. Both of these inhibitors had IC50 values above our screening maximum thresholds of 1000 µM for NI and 250 µM for HI (Figure 3). Neu5Ac2en (2) was relatively more potent, with NI and HI IC50 values of 63.8 µM and 15.9 µM, respectively. The most potent inhibition within this group was observed for inhibitor 9 ACS Paragon Plus Environment


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1 (the C-5 acetamido analogue of the potent hPIV-1 inhibitor, 5), with IC50 values of 3.92 µM for NI and 3.50 µM for HI. We observed a significant improvement in the inhibition of both HN functions for the C-5 isobutyramido series (5-7), relative to their C5 acetamido analogues, as demonstrated by the determined NI and HI IC50 values. However, the inhibition found with the zanamivir analogue 8 was still too weak to determine IC50 values within our assays detection limits. The calculated NI and HI IC50 values of 260.8 µM and 7.29 µM, respectively for inhibitor 7 were remarkably improved (compared with inhibitor 3). Following the same pattern observed in the C-5 acetamido series, inhibitor 6 (the C-5 analogue of Neu5Ac2en) was next best, with NI and HI IC50 values of 3.82 µM and 0.49 µM, respectively. Finally, IC50 values of 0.50 µM and 0.08 µM for NI and HI, respectively were determined for the potent reference inhibitor, 5. The key C-4 functionality features that have impact on inhibitor potency are likely to be size and charge related. The least favored C-4 functionality among the 4 evaluated pairs was the positively-charged guanidine group associated the weakest inhibitors 4 and 8. From inspection of the hPIV-1 homology model, the observed extremely weak inhibition of these two compounds can be rationalized on the basis of the hydrophobic nature of the C-4 binding domain of the hPIV-1 HN protein’s active site. Such a hydrophobic domain would not accommodate a bulky charged moiety such as a guanidine group. Interestingly, and consistent with this notion, the less bulky and less basic 4-amino group in 3 and 7 had relatively improved IC50 values when compared with the corresponding guanidine analogues. The 4hydroxyl group of the naturally-occurring inhibitor Neu5Ac2en (2) was indeed more favored when compared with either the amino or guanidine analogues. The best-accommodated C-4 functionality in both series, that is 1 and the reference inhibitor 5, was an azide group. A comparison of the IC50 values of these four pairs of inhibitors demonstrates a critical effect of the C-5 substituent on hPIV-1 HN inhibition. In all inhibitor pairs except (4) and (8), due to above threshold and 10 ACS Paragon Plus Environment

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weak inhibition, the improvement of inhibitor potency as a consequence of this replacement ranged from 8 ~ 17 fold for NI, and 32 ~ 44 fold for HI. The effect of the C-5 acylamino functionality on inhibitor potency was also investigated. Thus, the NI and HI IC50 values of the C-5 acylamino-Neu2en derivatives 22a-e and 24 were determined (Figure 4). Inhibitor 22a incorporates a cyclopropanamido functionality at C-5 in place of the isobutyramido group of 5 and provided an opportunity to explore the effect of planarity and ring strain on HN inhibition. The compound was found to be slightly less potent than 5 with NI and HI IC50 values of 0.91 µM and 0.32 µM, respectively and suggests that the C-5 binding domain can accommodate strained ring systems. Increasing the size of the ring from 3 carbons (22a) to 4 carbons (22b) led to a reduction in potency, with determined IC50 values of 1.92 µM and 0.93 µM for NI and HI, respectively.

Figure 4. The NI (plain) and HI (strips) IC50 values of the inhibitors 22a-e and 24. Extending the length of the C5-acylamino carbon chain, through addition of an extra carbon to the isobutyramido group (22c), also led to a decrease in potency with calculated NI and HI IC50 values of 1.58 µM and 0.32 µM, respectively. Restricting the free rotation of the acyl group of 22c through incorporation of a double bond (22d) resulted in an additional loss in potency, where NI and HI IC50 values were determined to be 7.76 µM and 3.0 µM, respectively. Interestingly, the replacement of one of the methyl groups of the isobutyramido functionality with a polar hydroxyl group (22e) led to a significant drop in potency. IC50 values of 68.1 µM and 40.0 µM for NI and HI, respectively were 11 ACS Paragon Plus Environment


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determined for 22e and consequently a 136 to 500 fold decrease in inhibition of HN functions was observed. This drop in inhibitor potency affirms the hydrophobic nature of the C-5 functional group binding pocket predicted from the homology model. To further investigate the effect of polar groups at C-5 on the Neu2en template we evaluated compound 24. This compound incorporates a terminal hydroxyl group on one of the isobutyramido methyl groups of 5. The compound was found to be less potent than the parent inhibitor 5 and the analogous inhibitor 22c. However, it was relatively more potent than 22e with NI and HI values of 9.49 µM and 3.0 µM, respectively. This outcome is consistent with our computational analysis of the hPIV-1 HN homology model and suggests that maintenance of the inhibitor’s hydrophobic interactions, derived from the methyl groups of 5, reduces significant loss of activity. Finally, potential cytotoxicity of the tested compounds was also evaluated by standard cell viability assays (see experimental and the Supporting information file for Supplementary Figures 1 & 2). In these assays, all of the tested compounds were found to be non-cytotoxic, with a minimum cell viability of 93% recorded for compound 6. CONCLUSION Our study demonstrates that the replacement of the Neu5Ac2en C-5 acetamido moiety with more hydrophobic alkane-based moieties, including an isobutyramido group, can improve inhibitory potency for both HN functions. Moreover, inhibitor potency was also impacted by characteristics, such as size and charge, of the C-4 functionality. These characteristics determine the inhibitor’s capacity to be appropriately accommodated within the Neu2en C-4 binding domain. Finally, our study provides new information towards the design of novel Neu2en-based hPIV-1 HN inhibitors and demonstrates that C-5 alkylamido groups are an optimal functionality for maximizing inhibitor potency.


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EXPERIMENTAL SECTION Computational Chemistry. hPIV-1 HN Homology Model Generation and Molecular Dynamics Simulations– MD simulations were performed with the GROMOS software27,28, using the force-field parameter set 54A4.29 To obtain the initial structure of hPIV-1 HN for MD, a homology model has been created based on the crystal structure of hPIV-3 HN30 (pdb: 1V3B). The sequence used to create the homology model is that of the strain C35, EMBL-bank number AB542810.1. A BLAST protein search showed the best match to an existing crystal structure was to the structure of hPIV-3 at 57% identical and 70% similar. The first 143 residues were not in the crystal structure and have been deleted. To create the homology model, the best resolution hPIV-3 HN structure 1V3B30 has been used. The alignment of the hPIV-1 HN sequence with the structure of hPIV-3 HN was done using the software Modeller 9v8.31 1000 models have been generated and the DOPE score was used to select the best model. Comparison of the model to the starting crystal structure has been done in SwissPDB Viewer32 and had an RMS fit of 0.02 nm. To place compounds 3 and 7 in the active site, the homology model of hPIV-1 HN has been superimposed with the X-ray structure of hPIV-3 HN in complex with 2-deoxy2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en, 2)30 (pdb: 1V3D). Compounds 3 and 7 have been superimposed over the ring atoms of Neu5Ac2en (2). Parameters for 3 and 7 have been generated in an analogous manner to existing parameters in the GROMOS forcefield. Ionization states of amino acid residues were assigned at pH 7.0. The histidine side chains were protonated at the Nε-atom. The simple-point-charge (SPC) water model33 was used to describe the solvent molecules. In the simulations, water molecules were added around the protein within a truncated octahedron with a minimum distance of 1.4 nm between the protein atoms and the square walls of the periodic box. All bonds were constrained with a geometric tolerance of 10−4 using the SHAKE algorithm.34 A steepestdescent energy minimization of the system was performed to relax the solute-solvent contacts, while positionally restraining the solute atoms using a harmonic interaction with a force constant of 2.5 x 104 kJ mol−1 nm−2. Next, steepest-descent energy minimization of the system without any restraints was 13 ACS Paragon Plus Environment


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performed to eliminate any residual strain. The energy minimizations were terminated when the energy change per step became smaller than 0.1 kJ mol−1. For the non-bonded interactions, a triple-range method with cutoff radii of 0.8/1.4 nm was used. Short-range van der Waals and electrostatic interactions were evaluated every time step based on a charge-group pair-list. Medium-range van der Waals and electrostatic interactions, between (charge group) pairs at a distance longer than 0.8 nm and shorter than 1.4 nm, were evaluated every fifth time step, at which point the pair list was updated. Outside the longer cutoff radius a reaction-field approximation35 was used with a relative dielectric permittivity of 78.5. The centre of mass motion of the whole system was removed every 1000 time steps. Solvent and solute were independently, weakly coupled to a temperature bath of 295 K with a relaxation time of 0.1 ps.36 The systems were also weakly coupled to a pressure bath of 1 atm with a relaxation time of 0.5 ps and an isothermal compressibility of 0.7513 x 10−3 (kJ mol−1nm−3)−1. 20 ps periods of MD simulation with harmonic position restraining of the solute atoms with force constants of 2.5 x 104 kJ mol−1 nm−2, 2.5 x 103 kJ mol−1 nm−2, 2.5 x 102 kJ mol−1 nm−2, 2.5 x 101 kJ mol−1 nm−2 were performed to equilibrate further the systems at 50 K, 120 K, 1800 K, 240 K and 300 K, respectively. MD simulations of hPIV-1 HN in complex with 3 and 7 were each carried out for 25 ns. The trajectory coordinates and energies were saved every 0.5 ps for analysis. Analysis– Analyses were done with the analysis software GROMOS++.37 Interaction energies between the HN protein and the inhibitor molecule have been calculated by extracting interaction energies from the energy trajectories of the simulations involving inhibitor molecules 3 and 7. Structural data for visual analysis has been extracted from the final frames of the coordinate trajectories. General Chemistry information. Reagents and dry solvents purchased from commercial sources were used without further purification. Anhydrous reactions were carried out under an atmosphere of argon, using oven-dried glassware. Reactions were monitored using thin layer chromatography (TLC) on aluminium plates pre-coated with Silica Gel 60 F254 (E. Merck). Developed plates were observed under 14 ACS Paragon Plus Environment

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UV light at 254 nm and then visualized after application of a solution of H2SO4 in EtOH (5% v/v) and heating. Flash chromatography was performed on Silica Gel 60 (0.040-0.063mm) using distilled solvents. 1H and

13

C NMR spectra were recorded either at 300 and 75.5 MHz respectively on a

BrukerAvance 300 MHz spectrometer or at 400 and 100 MHz respectively on a BrukerAvance 400 MHz spectrometer. Chemical shifts (δ) are reported in parts per million (ppm), relative to the residual solvent peak as internal reference [CDCl3: 7.26 (s) for 1H, 77.0 (t) for (pent) for 1H, 49.15 (hept) for 1

13

C; MeOD: 4.78 (s) and 3.31

13

C; DMSO: 2.50 (pent) for 1H, 39.51 (hept) for

13

C; D2O: 4.79 (s) for

H]. 2D COSY and HSQC experiments were run to support assignments. Low-resolution mass spectra

(LRMS) were recorded, in electrospray ionization mode, on a BrukerDaltonics Esquire 3000 ESI spectrometer, using positive mode. High-resolution mass spectrometry (HRMS) were recorded for either the protected or deprotected final derivatives, and were carried out by the University of Queensland FTMS Facility on a BrukerDaltonics Apex III 4.7e Fourier Transform micrOTOF-Q70 MS or by the Griffith University FTMS Facility on a BrukerDaltonics Apex III 4.7e Fourier Transform MS, fitted with an Apollo ESI source. Final deprotected sialic acid derivatives were purified over a GracePureTM SPE C18-Aq 5000 mg/20 mL column using 2% acetonitrile/H2O. The purities of all synthetic intermediates after chromatographic purification were judged to be >90% while the purities of the final products 1-8, 23a-e and 25 were judged to be ≥95% by HPLC analysis. Analytical HPLC was carried out on a Synergi Hydro-RP Phenomenex column (250 × 4.6 mm) eluted at a flow rate of 1.0 mL/min. An isocratic elution was performed with the suitable eluent and measurements were made at the wavelength corresponding to the maximum absorbance by each compound. The synthesis of reference inhibitors 1-4 followed typical procedures to that mentioned in the literature.20,22,38,39 Experimental procedures, analytical data and HRMS of new compounds are provided in the experimental section, while the analytical data and HRMS of reference inhibitors 1-4 and the NMR spectra of all the compounds are provided in the Supporting Information file.



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General procedure for the synthesis of 11 & 12. A mixture of 9 or 10 (0.42 mmol), Boc2O (275 mg, 1.27 mmol) and DMAP (50 mg, 0.42 mmol) in anhydrous THF (5 mL) was stirred under argon atmosphere at 60 °C o/n. After cooling to rt, the solvent was evaporated under vacuum, and the reside was taken in DCM for chromatographic separation on a silica gel column using ethyl acetate:hexane (1:2) to yield pure 11 (225 mg, 96%) or 12 (170 mg, 71%). Methyl

5-(N-acetyl-N-tert-butoxycarbonyl-amino)-7,8,9-tri-O-acetyl-2,6-anhydro-4-azido-3,4,5-

trideoxy-D-glycero-D-galacto-non-2-enonate (11).20 1H NMR (300 MHz, d6-DMSO): δ 1.52 (s, 9H, tBoc-3CH3), 1.96 (s, 6H, 2OAc), 1.97 (s, 3H, OAc), 2.33 (s, 3H, NAc), 3.72 (s, 3H, COOCH3), 4.05 (m, 1H, H-9), 4.47 (m, 1H, H-9’), 4.63–4.91 (m, 3H, H-4, H-5, H-6), 5.11–5.38 (m, 2H, H-7, H-8), 5.98 (d, J = 2.4 Hz, 1H, H-3); 13C NMR (75 MHz, d6-DMSO): δ 20.36, 20.42 (3 OCOCH3, NCOCH3), 27.20 (tBoc-3CH3), 50.32 (C-5), 52.47 (COOCH3), 56.93 (C-4), 61.08 (C-9), 65.92 (C-7), 69.56 (C-8), 76.54 (C-6), 85.44 (t-Boc-CCH3), 108.64 (C-3), 145.51 (C-2), 151.18 (t-Boc-OCO), 161.00 (COOCH3), 169.24, 169.69, 170.05 (3 OCOCH3), 172.71 (CH3CONH); LRMS [C23H32N4O12] (m/z): (+ve ion mode) 579.3 [M+Na]+; HRMS (API) (m/z): [M+Na]+calcd for C23H32N4NaO12 [M+Na]+ 579.1914; found, 579.1909. Methyl 5-(N-acetyl-N-tert-butoxycarbonyl-amino)-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxyD-glycero-D-galacto-non-2-enonate

(12).20,23 1H NMR (300 MHz, CDCl3): δ 1.55 (s, 9H, t-Boc-3CH3),

1.97 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.35 (s, 3H, NAc), 3.77 (s, 3H, COOCH3), 4.12 (dd, J = 12.4, 6.0 Hz, 1H, H-9), 4.59 (d, J = 12.7 Hz, 1H, H-9’), 4.75–5.40 (m, 4H, H-5, H-6, H-7, H-8), 5.89 (d, J = 2.7 Hz, 1H, H-3), 6.02 (d, J = 7.9 Hz, 1H, H-4); 13C NMR (75 MHz, d6-DMSO): δ 20.32, 20.38, 20.41, 20.45 (4 OCOCH3, NCOCH3), 27.28 (t-Boc-3CH3), 49.99 (C-5), 52.42 (COOCH3), 61.16 (C-9), 66.00 (C-4), 66.88 (C-7), 69.66 (C-8), 76.22 (C-6), 84.85 (t-Boc-CCH3), 109.34 (C-3), 145.65 (C-2), 154.37 (t-Boc-OCO), 161.05 (COOCH3), 169.29, 169.63, 170.06, 170.19 (4


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OCOCH3, CH3CONH); LRMS [C25H35NO14] (m/z): (+ve ion mode) 596.2 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C25H35NNaO14 [M+Na]+ 596.1955; found, 596.1950. General procedure for the synthesis of 13 & 14. To a methanolic solution of NaOMe, freshly prepared by dissolving sodium metal (0.39 mmol, 9 mg) in anhydrous MeOH (5 mL), was added compound 11 or 12 (0.26 mmol). The mixture was stirred at rt for 1h, then quenched with Amberlite® IR-120 (H+) resin (to pH = 5). The resin was filtered off, washed with MeOH (5 mL × 3) and the combined filtrate and washings were evaporated under vacuum. The residue was re-dissolved in pyridine (2 mL), and to the resulted solution was added acetic anhydride (0.5 mL). The reaction mixture was stirred at rt under argon atmosphere o/n, then the solvent and excess Ac2O were removed under vacuum. The residue was then taken in DCM for chromatographic separation on a silica gel column, using ethyl acetate:hexane (1:2) to yield pure 13 (84 mg, 63%) or 14 (112 mg, 81%). Methyl

7,8,9-tri-O-acetyl-2,6-anhydro-5-(N-tert-butoxycarboxamido)-4-azido-3,4,5-trideoxy-D-

glycero-D-galacto-non-2-enonate (13).20 1H NMR (300 MHz, CDCl3): δ 1.43 (s, 9H, t-Boc-3CH3), 2.05 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.13 (s, 3H, OAc), 3.68 (q, J = 9.3 Hz, 1H, H-5), 3.80 (s, 3H, COOCH3), 4.17 (dd, J = 12.4, 6.7 Hz, 1H, H-9), 4.28–4.42 (m, 2H, H-4, NH), 4.62 (dd, J = 12.4, 2.9 Hz, 1H, H-9’), 4.81 (d, J = 9.3 Hz, 1H, H-6), 5.33 (ddd, J = 6.7, 5.3, 2.9 Hz, 1H, H-8), 5.50 (dd, J = 5.4, 2.5 Hz, 1H, H-7), 5.95 (d, J = 2.7 Hz, 1H, H-3);

13

C NMR (75 MHz, CDCl3): δ 21.27, 21.41 (3

OCOCH3), 28.76 (t-Boc-3CH3), 49.80 (C-5), 53.11 (COOCH3), 58.94 (C-4), 62.58 (C-9), 68.31 (C-7), 71.31 (C-8), 76.74 (C-6), 81.36 (t-Boc-CCH3), 108.18 (C-3), 145.64 (C-2), 155.28 (t-Boc-OCO), 162.08 (COOCH3), 170.56, 170.61, 171.15 (3 OCOCH3); LRMS [C21H30N4O11] (m/z): (+ve ion mode) 537.3 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C21H30N4NaO11 [M+Na]+ 537.1809; found, 537.1803. Methyl 4,7,8,9-tetra-O-acetyl-2,6-anhydro-5-(N-tert-butoxycarboxamido)-3,5-dideoxy-D-glycero-Dgalacto-non-2-enonate (14).23 1H NMR (300 MHz, CDCl3): δ 1.39 (s, 9H, t-Boc-3CH3), 2.04 (s, 6H, 2 OAc), 2.06 (s, 3H, OAc), 2.11 (s, 3H, OAc), 3.78 (s, 3H, COOCH3), 4.01–4.12 (m, 1H, H-5), 4.17 (dd, 17 ACS Paragon Plus Environment


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J = 12.2, 6.8 Hz, 1H, H-9), 4.32 (dd, J = 9.0, 3.7 Hz, 1H, H-6), 4.58 (dd, J = 12.3, 3.4 Hz, 1H, H-9’), 4.65 (d, J = 9.8 Hz, 1H, NH), 5.35 (ddd, J = 6.8, 5.2, 3.5 Hz, 1H, H-8), 5.46 (dd, J = 7.5, 3.1 Hz, 1H, H4), 5.53 (t, J = 4.2 Hz, 1H, H-7), 5.97 (d, J = 3.0 Hz, 1H, H-3); 13C NMR (75 MHz, CDCl3): δ 20.67, 20.71, 20.78, 20.83 (4 OCOCH3), 28.14 (t-Boc-3CH3), 47.88 (C-5), 52.53 (COOCH3), 61.96 (C-9), 67.79 (C-7), 68.46 (C-4), 70.45 (C-8), 76.80 (C-6), 80.45 (t-Boc-CCH3), 107.97 (C-3), 145.01 (C-2), 154.84 (t-Boc-OCO), 161.64 (COOCH3), 169.78, 169.93, 170.56, 170.60 (4 OCOCH3); LRMS [C23H33NO13] (m/z): (+ve ion mode) 554.2 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C23H33NNaO13 [M+Na]+ 554.1850; found, 554.1844. General procedure for the synthesis of 15 & 16. To a solution of 13 or 14 (0.15 mmol) in anhydrous DCM (2 mL) was added TFA (230 µL, 3.0 mmol) and the mixture was stirred at rt under argon o/n. The reaction was diluted with DCM (20 mL) and quenched with sat. aq. NaHCO3 solution (20 mL). The DCM layer was washed with water, brine then dried over anhydrous Na2SO4. The dried organic solvent was concentrated under vacuum, and purified by silica gel chromatography using the suitable solvent system to yield pure 15 (53 mg, 85%) or 16 (58 mg, 90%). Methyl 7,8,9-tri-O-acetyl-5-amino-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2enonate (15).20 Silica gel column chromatography was run using ethyl acetate:hexane (2:1). 1H NMR (300 MHz, CD3CN): δ 1.99 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.71 (t, J = 9.5 Hz, 1H, H-5), 3.74 (s, 3H, COOCH3), 3.94–4.02 (m, 2H, H-4, H-6), 4.19 (dd, J = 12.5, 5.5 Hz, 1H, H-9), 4.45 (dd, J = 12.6, 2.7 Hz, 1H, H-9’), 5.31 (ddd, J = 7.8, 5.4, 2.7 Hz, 1H, H-8), 5.57 (d, J = 7.3 Hz, 1H, H-7), 5.87 (d, J = 2.5 Hz, 1H, H-3); 13C NMR (75 MHz, CD3CN): δ 21.28, 21.37, 21.47 (3 OCOCH3), 50.84 (C-5), 53.39 (COOCH3), 62.53 (C-4), 63.18 (C-9), 69.03 (C-7), 70.68 (C-8), 79.72 (C-6), 109.41 (C-3), 146.29 (C-2), 163.16 (COOCH3), 171.03, 171.76, 171.97 (3 OCOCH3); LRMS [C16H22N4O9] (m/z): (+ve ion mode) 437.1 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C16H22N4NaO9 [M+Na]+ 437.1284; found, 437.1279. 18 ACS Paragon Plus Environment

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Methyl


4,7,8,9-tetra-O-acetyl-5-amino-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-

enonate (16). Silica gel column chromatography was run using ethyl acetate:hexane (5:1). 1H NMR (300 MHz, CDCl3): δ 1.65 (s, 2H, NH2), 2.03 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.10 (s, 3H, OAc), 2.14 (s, 3H, OAc), 2.95 (t, J = 9.0 Hz, 1H, H-5), 3.76 (s, 3H, COOCH3), 3.95 (d, J = 9.8 Hz, 1H, H-6), 4.24 (dd, J = 12.6, 5.2 Hz, 1H, H-9), 4.54 (dd, J = 12.3, 2.5 Hz, 1H, H-9’), 5.27 (dd, J = 8.0, 2.7 Hz, 1H, H4), 5.42 (m, 1H, H-8), 5.55 (d, J = 6.4 Hz, 1H, H-7), 5.90 (s, 1H, H-3); 13C NMR (75 MHz, d6-DMSO): δ 25.65, 25.69, 25.77, 26.02 (4 OCOCH3), 52.82 (C-5), 57.52 (COOCH3), 66.82 (C-9), 72.76 (C-7), 74.40 (C-4), 76.37 (C-8), 82.98 (C-6), 113.50 (C-3), 149.37 (C-2), 166.70 (COOCH3), 174.68, 175.35, 175.40, 175.84 (4 OCOCH3); LRMS [C18H25NO11] (m/z): (+ve ion mode) 454.1 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C18H25NNaO11 [M+Na]+ 454.1325; found, 454.1320. General procedure for the synthesis of 17 & 18. To a solution of 15 or 16 (0.116 mmol) in DCM (2 mL) under argon was added Et3N (82 µL, 0.58 mmol) and isobutyryl chloride (18 µL, 0.17 mmol). The mixture was stirred at rt for 4 h, then loaded onto a silica gel column for chromatographic separation using ethyl acetate:hexane (1:1) to yield pure 17 (51 mg, 91%) or 18 (50 mg, 84%). Methyl

7,8,9-tri-O-acetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-5-isobutyramido-D-glycero-D-

galacto-non-2-enonate (17).23 1H NMR (300 MHz, CDCl3): δ 1.09–1.18 (m, 6H, isobutyryl-2CH3), 2.01 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.34 (m, 1H, isobutyryl-CH), 3.61–3.85 (m, 4H, COOCH3, H-5), 4.17 (dd, J = 12.5, 6.3 Hz, 1H, H-9), 4.50–4.69 (m, 3H, H-4, H-6, H-9’), 5.27 (m, 1H, H-8), 5.38 (dd, J = 5.6, 2.1 Hz, 1H, H-7), 5.92 (d, J = 2.6 Hz, 1H, H-3), 6.04 (d, J = 8.4 Hz, 1H, NH); 13C NMR (75 MHz, CDCl3): δ 18.95, 19.32 (isobutyryl-2CH3), 20.67, 20.73, 20.81 (3 OCOCH3), 35.74 (isobutyryl-CH), 49.14 (C-5), 52.53 (COOCH3), 57.33 (C-4), 61.91 (C-9), 67.68 (C-7), 70.63 (C8), 75.29 (C-6), 107.73 (C-3), 145.03 (C-2), 161.57 (COOCH3), 170.06, 170.35, 170.58 (3 OCOCH3), 177.59 (isobutyryl-CO); LRMS [C20H28N4O10] (m/z): (+ve ion mode) 507.1 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C20H28N4NaO10 [M+Na]+ 507.1703; found, 507.1698. 19 ACS Paragon Plus Environment


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Methyl 4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-5-isobutyramido-D-glycero-D-galacto-non2-enonate (18). 1H NMR (300 MHz, CDCl3): δ 1.10 (d, J = 6.8 Hz, 6H, isobutyryl-2CH3), 2.04 (s, 3H, OAc), 2.06 (s, 6H, 2OAc), 2.12 (s, 3H, OAc), 2.27 (m, 1H, isobutyryl-CH), 3.80 (s, 3H, COOCH3), 4.19 (dd, J = 12.3, 6.9 Hz, 1H, H-9), 4.30–4.47 (m, 2H, H-5, H-6), 4.60 (dd, J = 12.3, 3.2 Hz, 1H, H-9’), 5.33 (m, 1H, H-8), 5.41–5.52 (m, 2H, H-7, NH), 5.57 (dd, J = 7.2, 3.1 Hz, 1H, H-4), 5.99 (d, J = 3.0 Hz, 1H, H-3);

13

C NMR (75 MHz, CDCl3): δ 18.92, 19.32 (isobutyryl-2CH3), 20.67, 20.70, 20.76, 20.83 (4

OCOCH3), 35.59 (isobutyryl-CH), 46.34 (C-5), 52.53 (COOCH3), 61.96 (C-9), 67.61 (C-7), 68.05 (C4), 71.04 (C-8), 76.68 (C-6), 108.11 (C-3), 145.09 (C-2), 161.60 (COOCH3), 170.02, 170.31, 170.54, 170.80 (4 OCOCH3), 176.94 (isobutyryl-CO); LRMS [C20H29NO11] (m/z): (+ve ion mode) 524.3 [M+Na]+. General procedure for the synthesis of 5 & 6. To a suspension of compound 17 or 18 (0.08 mmol) in a (1:1) mixture of MeOH and water (2 mL) at 0 °C was added NaOH solution (1.0 M) dropwise until the pH reaches 13-14. The temperature was raised gradually to rt and the mixture was stirred at rt overnight. The solution was then acidified with Amberlite® IR-120 (H+) resin (to pH = 5), filtered and washed with MeOH (10 mL) and H2O (10 mL). The combined filtrate and washings were then concentrated under vacuum and the residue was diluted with distilled water (5 mL) and adjusted to pH = 8.0 using 0.05 M NaOH to convert the compound to its Na-salt. The compound was then purified over a C18-GracePureTM column, using 2% acetonitrile/water, to yield pure 5 (24 mg, 82%) or 6 (26 mg, 94%) as a fluffy white powder after freeze drying. Sodium

2,6-anhydro-4-azido-3,4,5-trideoxy-5-isobutyramido-D-glycero-D-galacto-non-2-enonate

(5).23 1H NMR (300 MHz, D2O): δ 1.14–1.17 (m, 6H, isobutyryl-2CH3), 2.58 (p, J = 6.9 Hz, 1H, isobutyryl-CH), 3.56–3.72 (m, 2H, H-7, H-9), 3.85–4.02 (m, 2H, H-8, H-9’), 4.22 (dd, J = 10.7, 9.2 Hz, 1H, H-5), 4.29–4.41 (m, 2H, H-4, H-6), 5.71 (d, J = 2.3 Hz, 1H, H-3);

13

C NMR (75 MHz, D2O): δ

18.45, 18.63 (isobutyryl-2CH3), 35.28 (isobutyryl-CH), 47.54 (C-5), 59.30 (C-4), 63.05 (C-9), 68.10 (C20 ACS Paragon Plus Environment

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7), 69.75 (C-8), 75.16 (C-6), 103.44 (C-3), 149.31 (C-2), 169.09 (COONa), 181.47 (isobutyryl-CO); LRMS [C13H19N4O7] (m/z): (+ve ion mode) 367.2 [M+Na]+. Purity by analytical HPLC (220 nm) = 96.5%, tR= 7.4 min (mobile phase: 22% acetonitrile/78% H2O containing 0.05% TFA). Sodium 2,6-anhydro-3,5-dideoxy-5-isobutyramido-D-glycero-D-galacto-non-2-enonate (6). 1H NMR (300 MHz, D2O): δ 1.14–1.17 (m, 6H, isobutyryl-2CH3), 2.59 (m, 1H, isobutyryl-CH), 3.57 (dd, J = 9.3, 1.2 Hz, 1H, H-7), 3.65 (dd, J = 11.8, 6.1 Hz, 1H, H-9), 3.85–4.00 (m, 2H, H-8, H-9’), 4.05 (dd, J = 10.9, 8.8 Hz, 1H, H-5), 4.25 (dd, J = 11.1, 1.2 Hz, 1H, H-6), 4.50 (dd, J = 8.8, 2.4 Hz, 1H, H-4), 5.71 (d, J = 2.3 Hz, 1H, H-3); 13C NMR (75 MHz, D2O): δ 18.46, 18.91 (isobutyryl-2CH3), 35.22 (isobutyryl-CH), 49.57 (C-5), 63.08 (C-9), 67.35 (C-4), 68.29 (C-7), 69.73 (C-8), 75.31 (C-6), 107.72 (C-3), 147.87 (C2), 169.65 (COONa), 181.78 (isobutyryl-CO); LRMS [C13H20NNaO8] (m/z): (+ve ion mode) 364.1 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C38H20NNa2O8 [M+Na]+ 364.0984; found, 364.0979. Purity by analytical HPLC (234 nm) = 99.7%, tR= 7.9 min (mobile phase: 2% acetonitrile/98% H2O containing 0.05% TFA). Synthesis of methyl 7,8,9-tri-O-acetyl-4-amino-2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-Dglycero-D-galacto-non-2-enonate (19). To a solution of intermediate 17 (200 mg, 0.41 mmol) in EtOH (5 mL) was added Lindlar catalyst (50 mg) and the mixture was stirred at rt o/n under H2 atmosphere. Upon reaction completion (monitored by TLC), palladium catalyst was filtered off over celite, and the celite bed was washed with EtOH (10 mL × 3). The combined filtrate and washings were evaporated under reduced pressure and the residue was taken in DCM and loaded to a silica gel column for chromatographic separation using ethyl acetate:methanol:water (10:1:0.5) to yield pure 19 (145 mg, 77%). 1H NMR (300 MHz, CDCl3): δ 1.12-1.16 (m, 6H, isobutyryl-2CH3), 2.03 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.10 (s, 3H, OAc), 2.35 (m, 1H, isobutyryl-CH), 3.65-3.84 (m, 5H, H-4, H-5, COOCH3), 4.18 (dd, J = 12.4, 7.0 Hz, 1H, H-9), 4.34 (dd, J = 9.6, 2.5 Hz, 1H, H-6), 4.66 (dd, J = 12.4, 2.8 Hz, 1H, H-9’), 5.29 (ddd, J= 7.5, 4.7, 2.9 Hz, 1H, H-8), 5.45 (dd, J = 4.8, 2.5 Hz, 1H, H-7), 5.71 (d, J = 9.1 Hz, 21 ACS Paragon Plus Environment


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1H, NH), 5.98 (d, J = 2.5 Hz, 1H, H-3);

13

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C NMR (75 MHz, CDCl3): δ 19.10, 19.64, 20.74, 20.90 (3

OCOCH3, isobutyryl-2CH3), 35.74 (isobutyryl-CH), 50.16 (C-5), 50.93 (C-4), 52.37 (COOCH3), 62.08 (C-9), 68.00 (C-7), 71.37 (C-8), 76.27 (C-6), 113.60 (C-3), 143.30 (C-2), 162.19 (COOCH3), 170.23, 170.37, 170.59 (3 OCOCH3), 177.62 (isobutyryl-CO); LRMS [C20H30N2O10] (m/z): (+ve ion mode) 481.1 [M+Na]+. Synthesis of sodium 4-amino-2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-D-glycero-D-galactonon-2-enonate (7). To a suspension of compound 19 (40 mg, 0.087 mmol) in a (1:1) mixture of MeOH and water (2 mL) at 0 °C was added NaOH solution (1 M) dropwise until the pH reaches 13-14. The temperature was raised gradually to rt and the mixture was stirred at rt overnight. The solution was then acidified with Amberlite@IR-120 (H+) resin (to pH = 5), filtered and washed with MeOH (10 mL) and H2O (10 mL). The combined filtrate and washings were then concentrated under vacuum and the residue was diluted with distilled water (5 mL) and adjusted to pH = 8.0 using 0.05 M NaOH to convert the compound to its Na-salt. The compound was then purified over a C18-GracePureTM column using 2% acetonitrile/water to yield after freeze drying 18 mg (61%) of 7 as a white fluffy powder. 1H NMR (300 MHz, D2O): δ 1.14-1.19 (m, 6H, isobutyryl-2CH3), 2.60 (m, 1H, isobutyryl-CH), 3.58–3.75 (m, 2H, H7, H-9), 3.91 (dd, J = 11.9, 2.7 Hz, 1H, H-9), 3.98 (ddd, J = 9.1, 6.0, 2.6 Hz, 1H, H-8), 4.22 (dt, J = 8.2, 1.9 Hz, 1H, H-4), 4.30–4.48 (m, 2H, H-5, H-6), 5.70 (d, J = 2.3 Hz, 1H, H-3);

13

C NMR (75

MHz, D2O): δ 18.02 (isobutyryl-CH3), 19.05 (isobutyryl-CH3), 35.02 (isobutyryl-CH), 45.76 (C-5), 49.94 (C-4), 62.95 (C-9), 67.92 (C-7), 69.62 (C-8), 74.99 (C-6), 99.94 (C-3), 150.63 (C-2), 168.49 (COONa), 181.70 (isobutyryl-CO); LRMS [C13H22N2O7] (m/z): (+ve ion mode) 341.1 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C13H22N2NaO7 [M+Na]+ 341,1319; found, 341.1325. Purity by analytical HPLC (234 nm) = 100%, tR= 4.8 min (mobile phase: 2% acetonitrile/98% H2O containing 0.05% TFA).


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Synthesis of methyl 7,8,9-tri-O-acetyl-2,6-anhydro-4-(N,N’-(di-tert-butyloxycarbonyl)guanidino)3,4,5-trideoxy-5-isobutyramido-D-glycero-D-galacto-non-2-enonate (20). A mixture of the amine 19 (100 mg, 0.22 mmol), N,N'-Di-Boc-1H-pyrazole-1-carboxamidine (136 mg, 0.44 mmol) and triethylamine (92 µL, 0.66 mmol) was stirred in anhydrous MeOH (2 mL) at rt for 40 h. The solvent was then removed under vacuum and the crude product was purified by silica gel chromatography using ethyl acetate:acetone (2:3) to yield 112 mg (73%) of pure 20. 1H NMR (300 MHz, CDCl3): δ 0.99-1.05 (m, 6H, isobutyryl-2CH3), 1.47 (s, 9H, t-butyl-3CH3), 1.48 (s, 9H, t-butyl-3CH3), 2.04 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.10 (s, 3H, OAc), 2.27 (m, J = 6.9 Hz, 1H, isobutyryl-CH), 3.77 (s, 3H, COOCH3), 4.06–4.41 (m, 3H, H-5, H-7, H-9), 4.71 (dd, J = 12.4, 2.6 Hz, 1H, H-6), 5.17 (td, J = 9.6, 2.5 Hz, 1H, H4), 5.26 (ddd, J = 7.3, 4.5, 2.6 Hz, 1H, H-8), 5.49 (dd, J = 4.6, 1.7 Hz, 1H, H-9’), 5.83 (d, J = 2.3 Hz, 1H, H-3), 6.37 (s, 1H, guanidine-NH), 7.29 (d, J = 9.4 Hz, 1H, NHAc), 8.50 (d, J = 9.4 Hz, 1H, C-4NH);

13

C NMR (75 MHz, CDCl3): δ 18.95, 19.12, 20.79, 20.81, 20.94 (3 OCOCH3, isobutyryl-2CH3),

28.03 (t-butyl-3CH3), 28.27 (t-butyl-3CH3), 35.72 (isobutyryl-CH), 47.10 (C-5), 48.87 (C-4), 52.42 (COOCH3), 62.37 (C-9), 67.77 (C-7), 71.92 (C-8), 78.00 (C-6), 80.00 (t-butyl q carbon), 83.92 (t-butyl q carbon), 109.82 (C-3), 145.10 (C-2), 152.61 (Guanidine C), 157.36 (t-butyl CO), 161.75 (t-butyl CO), 162.84 (COOCH3), 170.06, 170.43, 170.58 (3 OCOCH3), 177.53 (isobutyryl-CO); LRMS [C31H48N4O14] (m/z): (+ve ion mode) 723.3 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C31H48N4NaO14 [M+Na]+ 723.3065; found, 723.3059. Synthesis of sodium 2,6-anhydro-3,4,5-trideoxy-4-guanidino-5-isobutyramido-D-glycero-D-galactonon-2-enonate (8). To a solution of 20 (100 mg, 0.143 mmol) in DCM (3 mL) was added TFA (0.5 mL) and the mixture was stirred at rt for 20 h. The reaction was quenched by addition of solid NaHCO3 until effervescence ceases, filtered over celite, washed with DCM (20 mL × 3) and concentrated under vacuum. To a suspension of the crude product (40 mg, 0.08 mmol) in a (1:1) mixture of MeOH and water (2 mL) at 0 °C was added NaOH solution (1 M) dropwise until the pH reaches 13-14. The temperature was raised gradually to rt and the mixture was stirred at rt overnight. The solution was then 23 ACS Paragon Plus Environment


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acidified with Amberlite@IR-120 (H+) resin (to pH = 5), filtered and washed with MeOH (10 mL) and H2O (10 mL). The combined filtrate and washings were then concentrated under vacuum and the residue was diluted with distilled water (5 mL) and adjusted to pH = 8.0 using 0.05 M NaOH to convert the compound to its Na-salt. The compound was then purified over a C18-GracePureTM column using 2% acetonitrile/water to yield after freeze drying 20 mg (61%) of 8 as a white fluffy powder. 1H NMR (300 MHz, D2O): δ 1.14 (d, J = 6.9 Hz, 6H, isobutyryl-2CH3), 2.57 (m, 1H, isobutyryl-CH), 3.65-3.71 (m, 2H, H-7, H-9), 3.83–4.05 (m, 2H, H-8, H-9’), 4.28 (m, 1H, H-5), 4.49 (d, J = 10.6 Hz, 1H, H-6), 4.56 (dd, J = 9.1, 2.6 Hz, 1H, H-4), 5.90 (d, J = 2.5 Hz, 1H, H-3); 13C NMR (75 MHz, D2O): δ 18.36 (isobutyryl-CH3), 18.53 (isobutyryl-CH3), 35.23 (isobutyryl-CH), 47.38 (C-5), 50.91 (C-4), 62.96 (C-9), 67.96 (C-7), 69.83 (C-8), 75.78 (C-6), 107.28 (C-3), 146.28 (C-2), 156.94 (Guanidine-C), 166.48 (COOH), 181.44 (isobutyryl-CO); LRMS [C14H24N4O7] (m/z): (+ve ion mode) 383.0 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C14H24N4NaO7[M+Na]+ 383.1537; found, 383.1540. Purity by analytical HPLC (234 nm) = 99%, tR= 11.8 min (mobile phase: 0.5% acetonitrile/99.5% H2O containing 0.05% TFA). General procedure for the synthesis of compounds 21a-e. To a solution of 15 (50 mg, 0.12 mmol) in DCM (2 mL) was added Et3N (84 µL, 0.6 mmol) and the corresponding acid chloride (0.18 mmol). The mixture was stirred at rt for 4 h, then upon reaction completion (monitored by TLC) it was loaded to silica gel column for chromatographic separation using the suitable solvent. Methyl

7,8,9-tri-O-acetyl-2,6-anhydro-4-azido-5-cyclopropanamido-3,4,5-trideoxy-D-glycero-D-

galacto-non-2-enonate (21a). Silica gel chromatography using ethyl acetate:hexane (1:1) yielded 47 mg (82%) of pure 21a. 1H NMR (300 MHz, CDCl3): δ 0.80 (dt, J = 7.6, 3.5 Hz, 2H, cyclopr-CH2), 0.97 (td, J = 4.4, 2.2 Hz, 2H, cyclopr-CH2), 1.36 (tt, J = 8.1, 4.5 Hz, 1H, cyclopr-CH), 2.05 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.12 (s, 3H, OAc), 3.80–3.90 (m, 4H, COOCH3, H-5), 4.19 (dd, J = 12.4, 6.5 Hz, 1H, H-9), 4.46–4.65 (m, 3H, H-4, H-6, H-9’), 5.33 (td, J = 5.8, 5.3, 3.0 Hz, 1H, H-8), 5.45 (dd, J = 5.3, 2.6 Hz, 24 ACS Paragon Plus Environment

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1H, H-7), 5.93 (d, J = 8.6 Hz, 1H, NH), 5.98 (d, J = 2.8 Hz, 1H, H-3); 13C NMR (75 MHz, CDCl3): δ 7.61 (cyclopr-CH2), 7.68 (cyclopr-CH2), 14.82 (cyclopr-CH), 20.72, 20.77, 20.88 (3 OCOCH3), 48.96 (C-5), 52.59 (COOCH3), 57.33 (C-4), 61.97 (C-9), 67.84 (C-7), 70.58 (C-8), 75.61 (C-6), 107.48 (C-3), 145.05 (C-2), 161.55 (COOCH3), 170.07, 170.30, 170.56 (3 OCOCH3), 174.02 (cyclopr-CO); LRMS [C20H26N4O10] (m/z): (+ve ion mode) 504.7 [M+Na]+. Methyl

7,8,9-tri-O-acetyl-2,6-anhydro-4-azido-5-cyclobutanamido-3,4,5-trideoxy-D-glycero-D-

galacto-non-2-enonate (21b). Silica gel chromatography using ethyl acetate:hexane (1:1) yielded 55 mg (93%) of pure 21b. 1H NMR (300 MHz, CDCl3): δ 1.82–1.99 (m, 2H, cyclobut-CH2), 2.03 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.10–2.20 (m, 5H, OAc, cyclobut-CH2), 2.23–2.31 (m, 2H, cyclobut-CH2), 3.00 (p, J = 8.5 Hz, 1H, cyclobut-CH), 3.69–3.79 (m, 4H, COOCH3, H-5), 4.19 (dd, J = 12.5, 6.3 Hz, 1H, H-9), 4.49–4.66 (m, 3H, H-4, H-6, H-9’), 5.30 (td, J = 6.0, 2.7 Hz, 1H, H-8), 5.40 (dd, J = 5.5, 2.3 Hz, 1H, H-7), 5.74 (d, J = 8.5 Hz, 1H, NH), 5.95 (d, J = 2.6 Hz, 1H, H-3); 13C NMR (75 MHz, CDCl3): δ 18.09 (cyclobut-CH2), 20.70, 20.78, 20.86 (3 OCOCH3), 24.91 (cyclobut-CH2), 25.14 (cyclobut-CH2), 39.79 (cyclobut-CH), 49.04 (C-5), 52.57 (COOCH3), 57.29 (C-4), 61.92 (C-9), 67.68 (C-7), 70.60 (C8), 75.36 (C-6), 107.61 (C-3), 145.07 (C-2), 161.55 (COOCH3), 170.08, 170.41, 170.56 (3 OCOCH3), 175.52 (cyclobut-CO); LRMS [C21H28N4O10] (m/z): (+ve ion mode) 519.1 [M+Na]+. Methyl 7,8,9-tri-O-acetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-5-(2-methylbutanamido)-D-glyceroD-galacto-non-2-enonate

(21c). Silica gel chromatography using ethyl acetate:hexane (1:1) yielded 45

mg (75%) of pure 21c. 1H NMR (300 MHz, CDCl3): δ 0.94 (td, J = 7.4, 5.4 Hz, 3H, CH3CH2), 1.15 (dd, J = 10.6, 6.9 Hz, 3H, CH3CH), 1.59–1.76 (m, 2H, CH2), 2.03 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.12– 2.19 (m, 4H, CH, OAc), 3.47 (ddd, J = 22.8, 17.6, 8.8 Hz, 1H, H-5), 3.80 (s, 3H, COOCH3), 4.22 (dd, J = 12.5, 5.2 Hz, 1H, H-9), 4.54 (dt, J = 12.7, 2.0 Hz, 1H, H-9’), 4.63–4.91 (m, 2H, H-4, H-6), 5.24–5.48 (m, 2H, H-7, H-8), 5.81 (d, J = 7.8 Hz, 1H, NH), 5.96 (d, J = 2.4 Hz, 1H, H-3);

13

C NMR (75 MHz,

CDCl3): δ 11.80 (CH3CH2), 17.17 (CH3CH), 20.66, 20.82, 20.84 (3 OCOCH3), 26.83 (CH2), 43.41 25 ACS Paragon Plus Environment


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(CH), 50.26 (C-5), 52.57 (COOCH3), 56.60 (C-4), 61.76 (C-9), 67.75 (C-7), 70.03 (C-8), 74.40 (C-6), 107.51 (C-3), 145.00 (C-2), 161.59 (COOCH3), 169.87, 170.48, 170.61 (3 OCOCH3), 177.39 (CONH); LRMS [C21H30N4O10] (m/z): (+ve ion mode) 521.2 [M+Na]+. Methyl

7,8,9-tri-O-acetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-5-(E-2-methylbut-2-enamido)-D-

glycero-D-galacto-non-2-enonate (21d). Silica gel chromatography using ethyl acetate:hexane (1:1) yielded 46 mg (77%) of pure 21d. 1H NMR (300 MHz, CDCl3): δ 1.76 (d, J = 6.8 Hz, 3H, =CH-CH3), 1.83 (s, 3H, =C-CH3), 2.02 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.13 (s, 3H, OAc), 3.72 (q, J = 8.9 Hz, 1H, H-5), 3.80 (s, 3H, COOCH3), 4.20 (dd, J = 12.4, 6.2 Hz, 1H, H-9), 4.56 (dd, J = 12.5, 2.8 Hz, 1H, H-9’), 4.65–4.73 (m, 2H, H-4, H-7), 5.34 (td, J = 6.0, 2.7 Hz, 1H, H-8), 5.40 (dd, J = 5.8, 2.3 Hz, 1H, H-6), 5.97 (d, J = 2.7 Hz, 1H, H-3), 6.07 (d, J = 8.2 Hz, 1H, NH), 6.41 (q, J = 6.4 Hz, 1H, =CH-CH3); 13C NMR (75 MHz, CDCl3): δ 12.27 (=CH-CH3), 14.03 (=C-CH3), 20.68, 20.78, 20.87 (3 OCOCH3), 49.54 (C-5), 52.57 (COOCH3), 56.89 (C-4), 61.98 (C-9), 67.87 (C-7), 70.37 (C-8), 75.20 (C-6), 107.60 (C-3), 131.58 (C=CH), 131.87 (C=CH), 145.00 (C-2), 161.59 (COOCH3), 169.78, 170.00, 170.45, 170.55 (3 OCOCH3, CONH); LRMS [C21H28N4O10] (m/z): (+ve ion mode) 518.8 [M+Na]+. Methyl

7,8,9-tri-O-acetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-5-(2-hydroxypropionamido)-D-

glycero-D-galacto-non-2-enonate (21e). Silica gel chromatography using ethyl acetate:hexane (3:2) yielded 50 mg (78%) of pure 21e. 1H NMR (300 MHz, CDCl3): δ 1.46 (d, J = 6.9 Hz, 3H, O-CH-CH3), 2.05 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.13 (s, 3H, OAc), 2.17 (s, 3H, OAc), 3.41 (q, J = 9.0 Hz, 1H, H5), 3.79 (s, 3H, COOCH3), 4.22 (dd, J = 12.5, 5.4 Hz, 1H, H-9), 4.52 (dd, J = 12.5, 2.6 Hz, 1H, H-9’), 4.67 (d, J = 10.4 Hz, 1H, H-6), 4.95 (dd, J = 9.0, 2.8 Hz, 1H, H-4), 5.12 (q, J = 6.9 Hz, 1H, O-CH-CH3), 5.23 (d, J = 6.8 Hz, 1H, H-7), 5.34 (td, J = 5.9, 2.6 Hz, 1H, H-8), 5.93 (d, J = 2.5 Hz, 1H, H-3), 6.62 (d, J = 7.7 Hz, 1H, NH); 13C NMR (75 MHz, CDCl3): δ 17.48 (O-CH-CH3), 20.66, 20.83, 20.88, 20.97 (4 OCOCH3), 50.43 (C-5), 52.58 (COOCH3), 56.09 (C-4), 61.65 (C-9), 67.90 (C-7), 69.84 (O-CH-CH3),


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70.60 (C-8), 73.98 (C-6), 107.43 (C-3), 144.91 (C-2), 161.52 (COOCH3), 169.90, 169.97, 170.65, 170.68, 171.51 (4 OCOCH3, CONH); LRMS [C21H28N4O12] (m/z): (+ve ion mode) 551.2 [M+Na]+. General procedure for the synthesis of compounds 22a-e. To a suspension of compound 21a-e (0.08 mmol) in a (1:1) mixture of MeOH and water (2 mL) at 0 ºC was added NaOH solution (1 M) dropwise until the pH reaches 13-14. The temperature was raised gradually to rt and the mixture was stirred at rt overnight. The solution was then acidified with Amberlite@IR-120 (H+) resin (to pH = 5), filtered and washed with MeOH (10 mL) and H2O (10 mL). The combined filtrate and washings were then concentrated under vacuum and the residue was diluted with distilled water (5 mL) and adjusted to pH = 8.0 using 0.05 M NaOH to convert the compound to its Na-salt. The compound was then purified over a C18-GracePureTM column using 2% acetonitrile/water to yield after freeze drying the pure product 22ae as a white fluffy powder. Sodium

2,6-anhydro-4-azido-5-cyclopropanamido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-

enonate (22a). 1H NMR (300 MHz, D2O): δ 0.84–1.00 (m, 4H, cyclopr-2CH2), 1.68 (tt, J = 6.8, 5.0 Hz, 1H, cyclopr-CH), 3.58–3.74 (m, 2H, H-7, H-9), 3.87–4.02 (m, 2H, H-8, H-9’), 4.17–4.46 (m, 3H, H-4, H-5, H-6), 5.71 (d, J = 2.3 Hz, 1H, H-3);

13

C NMR (75 MHz, D2O): δ 6.99 (cyclopr-2CH2), 14.20

(cyclopr-CH), 47.92 (C-5), 59.37 (C-4), 63.12 (C-9), 68.05 (C-7), 69.77 (C-8), 75.29 (C-6), 103.44 (C3), 149.31 (C-2), 169.09 (COONa), 177.75 (cyclopr-CO); LRMS [C13H17N4NaO7] (m/z): (+ve ion mode) 387.0 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C13H17N4Na2O7 [M+Na]+ 387.0893; found, 387.0887. Purity by analytical HPLC (242 nm) = 98.7%, tR= 16.5 min (mobile phase: 10% acetonitrile/90% H2O containing 0.05% TFA). Sodium 2,6-anhydro-4-azido-5-cyclobutanamido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate (22b). 1H NMR (300 MHz, D2O): δ 1.84 (qd, J = 12.0, 9.9, 4.5 Hz, 1H, cyclobut-CH2), 2.01 (tt, J = 17.9, 8.7 Hz, 1H, cyclobut-CH2), 2.15–2.29 (m, 4H, cyclobut-2CH2), 3.25 (p, J = 8.6 Hz, 1H, cyclobutCH), 3.53–3.73 (m, 2H, H-7, H-9), 3.83–4.04 (m, 2H, H-8, H-9’), 4.15–4.43 (m, 3H, H-4, H-5, H-6), 27 ACS Paragon Plus Environment


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5.70 (d, J = 2.0 Hz, 1H, H-3);

13

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C NMR (75 MHz, D2O): δ 17.49(cyclobut-CH2), 24.82 (cyclobut-

2CH2), 39.28 (cyclobut-CH), 47.68 (C-5), 59.27 (C-4), 63.07 (C-9), 68.06 (C-7), 69.73 (C-8), 75.16 (C6), 103.46 (C-3), 149.29 (C-2), 169.08 (COONa), 179.13 (cyclobut-CO); LRMS [C14H19N4NaO7] (m/z): (+ve ion mode) 401.0 [M+Na]+; HRMS (API) (m/z): [M+H]+ calcd for C14H20N4NaO7 [M+H]+ 379.1230; found, 379.1224. Purity by analytical HPLC (242 nm) = 98.5%, tR= 21.7 min (mobile phase: 13% acetonitrile/87% H2O containing 0.05% TFA). Sodium

2,6-anhydro-4-azido-3,4,5-trideoxy-5-(2-methylbutanamido)-D-glycero-D-galacto-non-2-

enonate (22c). 1H NMR (400 MHz, CD3OD): δ 0.98 (t, J = 7.4 Hz, 3H, CH3CH2), 1.20 (d, J = 6.8 Hz, 3H, CH3CH), 1.49 (m, 1H, CH3CH2), 1.72 (m, 1H, CH3CH2), 2.34 (m, 1H, CH3CH), 3.60–3.71 (m, 2H, H-7, H-9), 3.86 (dd, J = 11.4, 2.9 Hz, 1H, H-9’), 3.93 (ddd, J = 8.9, 5.4, 3.0 Hz, 1H, H-8), 4.16 (m, 1H, H-5), 4.32 (d, J = 10.8 Hz, 1H, H-6), 4.42 (dd, J = 9.4, 2.2 Hz, 1H, H-4), 5.94 (d, J = 2.3 Hz, 1H, H-3); 13

C NMR (75 MHz, D2O): δ 11.25 (CH3CH2), 16.67 (CH3CH), 26.73 (CH3CH2), 42.86 (CH3CH), 47.52

(C-5), 59.38 (C-4), 63.05 (C-9), 68.17 (C-7), 69.75 (C-8), 75.09 (C-6), 103.52 (C-3), 149.26 (C-2), 169.01 (COONa), 180.90 (NH-CO); LRMS [C14H21N4NaO7] (m/z): (+ve ion mode) 403.0 [M+Na]+; HRMS (API) (m/z): [M+H]+calcd for C14H22N4NaO7 [M+H]+ 381.1386; found, 381.1381. Purity by analytical HPLC (245 nm) = 98.6%, tR= 29.3 min (mobile phase: 13% acetonitrile/87% H2O containing 0.05% TFA). Sodium

2,6-anhydro-4-azido-3,4,5-trideoxy-5-(E-2-methylbut-2-enamido)-D-glycero-D-galacto-

non-2-enonate (22d). 1H NMR (300 MHz, D2O): δ 1.80 (d, J = 6.9 Hz, 3H, =CH-CH3), 1.86 (s, 3H, CH3), 3.61–3.67 (m, 2H, H-7, H-9), 3.83–4.04 (m, 2H, H-8, H-9), 4.24–4.49 (m, 3H, H-4, H-5, H-6), 5.72 (d, J = 2.0 Hz, 1H, H-3), 6.47 (q, J = 6.9, 6.4 Hz, 1H, =CH-CH3);

13

C NMR (75 MHz, D2O): δ

11.63 (=CH-CH3), 13.31 (CH3), 48.01 (C-5), 59.29 (C-4), 63.10 (C-9), 68.16 (C-7), 69.76 (C-8), 75.25 (C-6), 103.47 (C-3), 130.70 (=C-CO), 133.81 (=CH-CH3), 149.37 (C-2), 169.12 (COONa), 173.46 (NHCO); LRMS [C14H19N4NaO7] (m/z): (+ve ion mode) 401.0 [M+Na]+; HRMS (API) (m/z): [M+H]+calcd 28 ACS Paragon Plus Environment

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for C14H20N4NaO7 [M+H]+ 379.1230; found, 379.1224. Purity by analytical HPLC (242 nm) = 97.5%, tR= 19.4 min (mobile phase: 11% acetonitrile/89% H2O containing 0.05% TFA). Sodium 2,6-anhydro-4-azido-3,4,5-trideoxy-5-(2-hydroxypropionamido)-D-glycero-D-galacto-non2-enonate (22e). 1H NMR (400 MHz, CD3OD): δ 1.43 (d, J = 6.8 Hz, 3H, CH3), 3.63 (d, J = 9.1 Hz, 1H, H-7), 3.69 (dd, J = 11.4, 5.3 Hz, 1H, H-9), 3.86 (dd, J = 11.5, 3.0 Hz, 1H, H-9’), 3.93 (ddd, J = 8.8, 5.5, 3.2 Hz, 1H, H-8), 4.14–4.29 (m, 2H, H-5, H-6), 4.41 (dd, J = 10.9, 1.1 Hz, 1H, CH), 4.52 (dd, J = 9.4, 2.1 Hz, 1H, H-4), 5.94 (d, J = 2.4 Hz, 1H, H-3);

13

C NMR (75 MHz, D2O): δ 19.66 (CH3), 47.47

(C-5), 59.11 (C-4), 63.04 (C-9), 67.79 (C-7), 68.04 (CHOH), 69.77 (C-8), 74.98 (C-6), 103.32 (C-3), 149.40 (C-2), 169.07 (COONa), 178.04 (NH-CO); LRMS [C12H17N4NaO8] (m/z): (+ve ion mode) 390.9 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C12H17N4Na2O8 [M+Na]+ 391.0842; found, 391.0836. Purity by analytical HPLC (243 nm) = 97.5%, tR= 12.4 min (mobile phase: 5% acetonitrile/95% H2O containing 0.05% TFA). Synthesis

of

methyl

7,8,9-tri-O-acetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-5-(3-hydroxy-2-

methylbutanamido)-D-glycero-D-galacto-non-2-enonate (23). A mixture of the amine 15 (50 mg, 0.12 mmol),

3-hydroxy-2-methylpropionic

acid

(12

µL,

0.12

mmol),

1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDCI) (69 mg, 0.36 mmol) and (1-hydroxybenzotriazole) HOBT (16 mg, 0.12 mmol) in DMF (2 mL) was stirred at rt, o/n under argon. The solvent was removed under vacuum, and the residue was purified by silica gel chromatography using ethyl acetate:hexane (2:1) to yield 38 mg (63%) of pure 23. 1H NMR (300 MHz, CDCl3): δ 1.07 (d, J = 6.7 Hz, 3H, CH3), 2.04 (s, 6H, 2OAc), 2.16 (s, 3H, OAc), 2.44 (m, 1H, CH), 3.55 (m, 1H, CH2), 3.71 (m, 1H, CH2), 3.80 (s, 3H,COOCH3), 4.11–4.23 (m, 2H, H-5, H-9), 4.28 (d, J = 9.4 Hz, 1H, H-4), 4.36 (d, J = 10.2 Hz, 1H, H9’), 4.71 (dd, J = 12.6, 2.6 Hz, 1H, H-6), 5.23 (m, 1H, H-8), 5.46 (d, J = 4.6 Hz, 1H, H-7), 5.95 (s, 1H, H-3), 6.62 (d, J = 9.2 Hz, 1H, NH); 13C NMR (75 MHz, CDCl3): δ 13.26 (CH3), 20.75, 20.90, 20.97 (3 OCOCH3), 43.86 (CH), 47.25 (C-5), 52.62 (COOCH3), 58.74 (C-4), 61.90 (C-9), 64.94 (CH2), 67.81 29 ACS Paragon Plus Environment


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(C-7), 71.62 (C-8), 76.28 (C-6), 107.73 (C-3), 145.16 (C-2), 161.48 (COOCH3), 170.63, 170.67, 171.54 (3 OCOCH3), 175.82 (NH-CO); LRMS [C20H28N4O11] (m/z): (+ve ion mode) 723.2 [M+Na]+. Synthesis of sodium 2,6-anhydro-4-azido-3,4,5-trideoxy-5-(3-hydroxy-2-methylbutanamido)-Dglycero-D-galacto-non-2-enonate (24). To a suspension of compound 23 (30 mg, 0.06 mmol) in a (1:1) mixture of MeOH and water (2 mL) at 0 ºC was added NaOH solution (1 M) dropwise until the pH reaches 13-14. The temperature was raised gradually to rt and the mixture was stirred at rt overnight. The solution was then acidified with Amberlite@IR-120 (H+) resin (to pH = 5), filtered and washed with MeOH (10 mL) and H2O (10 mL). The combined filtrate and washings were then concentrated under vacuum and the residue was diluted with distilled water (5 mL) and adjusted to pH = 8.0 using 0.05 M NaOH to convert the compound to its Na-salt. The compound was then purified over a C18GracePureTM column using 2% acetonitrile/water to yield after freeze drying 17 mg (72%) of the pure product 24 as a white fluffy powder. 1H NMR (300 MHz, D2O): δ 1.13 (d, J = 7.0 Hz, 3H, CH3), 2.68 (m, 1H, CH), 3.54–3.80 (m, 4H, CH2, H-7, H-9), 3.84–4.03 (m, 2H, H-8, H-9’), 4.21–4.42 (m, 3H, H-4, H-5, H-6), 5.72 (d, J = 2.2 Hz, 1H, H-3); 13C NMR (75 MHz, D2O): δ 12.97 (CH3), 43.34 (CH), 47.62 (C-5), 59.33 (C-4), 63.12 (C-9), 63.71 (CH2), 67.99 (C-7), 69.79 (C-8), 75.15 (C-6), 103.42 (C-3), 149.32 (C-2), 169.07 (COONa), 178.48 (NH-CO); LRMS [C13H19N4NaO8] (m/z): (+ve ion mode) 405.0 [M+Na]+; HRMS (API) (m/z): [M+Na]+ calcd for C13H19N4Na2O8 [M+Na]+ 405.0998; found, 405.0993. Purity by analytical HPLC (240 nm) = 97.1%, tR= 32.7 min (mobile phase: 5% acetonitrile/95% H2O containing 0.05% TFA). Biological Screening. Cells and virus– LLC-MK2 cells (kidney, Rhesus monkey) were used for hPIV-1 stock amplification. Cells were grown in Eagle's minimal essential medium (EMEM) (Lonza, Basel, Switzerland) supplemented with 1% Glutamine (200 mM) and 2% of fetal bovine serum (EMEMFBS) at 37 °C in a humidified atmosphere of 5% CO2.


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hPIV-1 (strain C-35) was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The virus was propagated in LLC-MK2 cells with EMEM supplemented with glutamine and 1µg/mL of trypsin TPCK (Sigma-Aldrich®, St. Louis, MO) (EMEMinf) at 35 ºC in a humidified atmosphere of 5% CO2. Virus-containing culture supernatant was collected 4 to 5 days post-infection, while monitoring cytopathic effects, and clarified from cell debris by centrifugation (4,500 RCF for 15 min). Virus was concentrated at least 10 times using 30 kDa Amicon Ultra filter unit (Millipore, Billerica, MA) for use in Hemagglutin Inhibition (HI) assays. In Neuraminidase Inhibition (NI) assays, virus was PEGprecipitated and then purified as described above. Clarified hPIV-1 supernatant was mixed with PEG6000 (8% final concentration) and NaCl (0.4 M final concentration) then incubated overnight at 4 ºC under gentle agitation. PEG6000/hPIV-1 complex was pelleted by centrifugation at 3,000 RCF for 30 min at 4 ºC. The supernatant was discarded and a volume of GNTE buffer (Glycine 200 mM, NaCl 200 mM, Tris-HCl 20 mM, EDTA 2 mM, pH 7.4) corresponding to at least 1:40 of the initial virus suspension volume was used to resuspend the pellet overnight at 4 ºC. The virus suspension was homogenized by up and down pipetting followed by a mechanical disruption of the remaining virus aggregates using a douncer with “tight” pestle. The hPIV-1 homogenate was loaded on top of a 30% 60% non-linear sucrose gradient prepared in GNTE buffer and centrifuged at 100,000 RCF for 2 h 30 min at 4 ºC without brake for deceleration. The virus was concentrated at the 30% - 60% sucrose interface that was collected and stored at -80 ºC. Neuraminidase inhibition assay– The purified hPIV-1 neuraminidase activity (NA) was assayed by the method of Potier et al24 with modificaiton13,25,26 and measured the relative fluorescence of 4methylumbelliferone,

the

product

of

the

neuraminidase

enzymatic

hydrolysis

of

2´-(4-

methylumbelliferyl) α-D-N-acetylneuraminide (MUN, Sigma-Aldrich, St Louis, MO). Purified hPIV-1, compounds and MUN were prepared and diluted in NA Reaction Buffer (NaOAc 50 mM, CaCl2 5 mM, pH 5.0). NA from different hPIV-1 dilutions were first measured to determined the lowest virus



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Page 32 of 39

concentration to be used for the assay. Indeed to be relevant, NA assay must be done with enough purified-virus to obtain a maximal signal at least 5 times higher than the background signal. Neuraminidase inhibition (NI) assay was done in triplicate. For each concentration tested, the wells received 2 µL of purified hPIV-1 and 4 µL of 2.5X HN inhibitor solution (1X final). The plate was kept at room temperature for 20 min before adding to each well 4 µL of 5 mM MUN (2 mM final). Plate was incubated a 37 ºC for 30 min under agitation (1100 rpm). The enzymatic reaction was stopped with the addition of 190 µL of Glycine buffer (Glycine 0.25 M, pH 10.4). A negative control was done by combining virus and MUN and stopping the enzymatic reaction at t=0. The relative fluorescence (RF) was measured with a Victor 3 multilabel reader (PerkinElmer, Waltham, MA). The data were processed by subtracting the background (negative control RF) and analysed with GraphPad Prism 4 (GraphPad Software Inc., La Jolla, CA) to calculate the compound’s concentration at NI IC50 (nonlinear regression (curve fit), Dose-response - inhibition, 3 parameter logistic). Hemagglutinination inhibition assay– Chicken red blood cells (C-RBC) were used in the hemagglutination inhibition (HI) assay.13 The HN inhibitors were assessed in duplicate in U-bottom 96 well plate. Compounds were diluted in PBS as a 4X solution for each concentration tested (25 µL/well, 1X final). Each dilution was mixed with 4 hemagglutination units (HAU) of hPIV-1 (25 µL/well, 1 HAU final) and incubated for 20 min at room temperature. The plate was transferred on ice and an equivalent volume of ice-cold 0.5% C-RBC (50 µL/well) was added to each well. Plate was incubated for 1 h and 30 min at 4 ºC before reading hemagglutination results. The IC50 value for HI assays was defined as the compound concentration that gives similar agglutination to that observed in a well containing solely 0.5 HAU of hPIV-1 and C-RBC. Cytotoxicity assay– Potential cytotoxicity of the tested compounds was evaluated by 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and AlamarBlue® cell viability assays.40 All of the inhibitors were tested in triplicate using LLC-MK2 cells for 48 h at a concentration of 250 µM (the maximum threshold for the HI assay). 32 ACS Paragon Plus Environment

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ASSOCIATED CONTENT Additional chemistry, biological assay figures illustrating cell viability data and NMR spectra are provided. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *

Prof

Mark

von

Itzstein,

Institute

for

Glycomics,

Griffith

University.

Email:

[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡ These authors contributed equally and are joint senior authors*. Funding Sources The project was supported by funding from the Australian Research Council, the National Health and Medical Research Council and Griffith University.

ACKNOWLEDGMENTS The Australian Research Council (DP1094549) is gratefully acknowledged for its financial support (MvI) and for the award of an Australian Postdoctoral Award (PG). The National Health and Medical Research Council (1047824) is thanked for its financial support (MvI). Griffith University is gratefully acknowledged for the award of a Griffith University Postdoctoral Award (IMED). ABBREVIATIONS hPIV(s), human parainfluenza virus(es); hPIV-1, human parainfluenza virus

type-1; HN,

hemagglutinin-neuraminidase; Neu5Ac2en, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid; Neu2en, 33 ACS Paragon Plus Environment


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2-deoxy-2,3-didehydro-neuraminic acid; MD, molecular dynamics; Boc, butyloxycarbonyl; DMAP, dimethylaminopyridine; THF, tetrahydrofuran; TFA, trifluoroacetic acid; DCM, dicholoromethane; DMF, N,N-dimethylformamide; NI, neuraminidase inhibition; HI, hemagglutinination inhibition; rt, room temperature; s, singlet; d, doublet; t, triplet; dd, doublet of doublet; ddd, doublet of doublet of doublet; dt, doublet of triplet; td; triplet of doublet; m, multiplet; TLC, thin layer chromatography; MUN, 2´-(4-methylumbelliferyl) α-D-N-acetylneuraminide.

REFERENCES (1) Karron, R. A.; Collins, P. L. Parainfluenza viruses. In Knipe, D. M.; Howley, P. M. (Editors). Fields Virology. 5th ed. Philadelphia: Lippincott/The Williams & Wilkins Co., a WoltersKluwersBuisness 2007; pp. 1497-1526. (2) Knott, A. M.; Long, C. E.; Hall, C. B. Parainfluenza viral infections in pediatric outpatients: seasonal patterns and clinical characteristics. Pediatr. Infect. Dis. J. 1994, 13, 269–273. (3) Marx, A.; Torok, T. J.; Holman, R. C.; Clarke, M. J.; Anderson, L. J. Pediatric hospitalization for croup (laryngotracheobronchitis): biennial increases associated with human parainfluenza virus 1 epidemics. J. Infect. Dis. 1997, 176, 1423–1427. (4) Reed, G.; Jewett, P. H.; Thompson, J.; Tollefson, S.; Wright, P. F. Epidemiology and clinical impact of parainfluenza virus infections in otherwise healthy infants and young children

Exploring Human Parainfluenza Virus Type-1 Hemagglutinin

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