Novel nucleic acid prodrugs and methods use thereof.

(54) Title: NOVEL NUCLEIC ACID DRUGS AND METHODS FOR USING THEM. (54) Title: NOVEL NUCLEIC ACID PRODRUGS AND METHODS USE THEREOF.

(57) Summary

The present invention relates to nucleic acid prodrugs and nucleic acid prodrugs comprising chiral phosphorous residues. Methods for preparing and using nucleic acid prodrugs and nucleic acid prodrugs comprising chiral phosphorous residues are also described herein .

(57) Abstract

Described herein are nucleic acid prodrugs and nucleic acid prodrugs comprising chiral phosphorous moieties. Also described herein are methods of making and using nucleic acid prodrugs and nucleic acid prodrugs comprising chiral phosphorous moieties.

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<img file="MX342945B_D0002.tif" />

Institute

Mexican Property

Industrial

PATENT TITLE NO. 342945 _SE_ seottwíA w K »wk

Owner (s): ONTORII, INC.

Address: 419 Western Avenue, Boston, Massachusetts, 02135, USA

Name: NOVEL NUCLEIC ACID DRUGS AND METHODS FOR USING THEM.

Classification:

Inventor (s):

Int.CI.8: A61K31 / 7052; A61K31 / 711; A61P35 / 00; C07H21 / 04 GREGORY L. VERDINE; MEENA MEENA; NAOKI IWAMOTO

REQUEST

Number:

MX / a / 2012/000380

Country:

US

US

Validity: Twenty years

International filing date:

July 2010

PRIORITY

Date:

July 2009 September 15, 2009

Number:

61/223,360

61/242,722

Expiration Date: July 6, 2030

The reference patent is granted based on articles 1, 2nd section V, 6th section III, and 59 of the Industrial Property Law.

In accordance with article 23 of the Industrial Property Law, this patent has a validity of twenty non-extendable years, counted from the date of filing of the international application and will be subject to the payment of the fee to keep the rights in force. .

Whoever signs this title does so based on the provisions of articles 6 'sections III and 7 ° bis 2 of the Industrial Property Law (Official Gazette of the Federation DOF) 08/27/1991, amended on 02 / 38/1994, 10/28/1996, 12/26/1997, 05/17/1999, 01/26/2004, 06/16/2005, 01/25/2006, 06/05/2009 / 06/01 / 2010, 06/18/2010, 06/28,2010, 01/27/2012 and 04/09/2012); Articles V, 3rd fraction V Clause a), 4th and 12th fractions I and III of the Regulations of the Mexican Institute of Prople &amp; d Industrial (DOF 14/12/1999, amended on 07/01/2002, 07/15/2004, 07/28/2004 and 09/07/2007); Articles 1, 3 * 4®, 5th fraction V subsection a), 16 fractions I and III and 30 of the Organic Statute of the Mexican Institute of Industrial Property (DOF 12/27/1999, amended on 10/10/2002, 07/29/2004, 08/04/2004 and 09/13/2007); 1, 3 and 5 subsection a) of the Agreement that delegates powers to the Deputy Directors General, Coordinator, Divisional Directors, Holders of the Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

<img file="MX342945B_D0003.tif" />

Issue Date: October 18, 2016

THE DIVISIONAL DIRECTOR OF PATENTS

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NAHANNY CANAL REYES

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<img file="MX342945B_D0006.tif" />

Arenal No. 550. Floor 1.

Coi. Pueblo Santa María Tepepan,

Xochimilco. CP 1SC20.

Mexico City on. (55) 53 34 07 00 www.impigob.mx

MX / 2016/84590

<img file="MX342945B_D0007.tif" />

IMÉÍ

MEXICAN INSTITUTE £&gt; £ LA EROEIEDAP - INDUSTRIAL

NOVEL NUCLEIC ACID DRUGS AND METHODS OF USE OF

THE SAME

FIELD OF THE INVENTION

Nucleic acid prodrugs and nucleic acid prodrugs comprising chiral phosphorous residues and methods of making and using them are described herein.

BACKGROUND OF THE INVENTION

Oligonucleotides are useful in new and nanomaterial applications, therapy, diagnostics, research and applications. The use of natural DNA or RNA sequences is limited, for example, by their stability to nucleases.

Additionally, in vitro studies show that antisense nucleotide properties such as binding affinity, specific binding of sequences to complementary RNA, stability to nucleases, are affected by the configurations of phosphorous atoms. Accordingly, there is a need for stereodefined oligonucleotide prodrugs to impart additional stability to oligonucleotide molecules in a number of in-vitro and in-vivo applications. Stereodefined oligonucleotide prodrugs comprising modified nucleic acids with and methods of use thereof are described herein.

<img file="MX342945B_D0008.tif" />

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL phosphorous atoms

SUMMARY OF THE INVENTION

One embodiment provides a chiral nucleic acid prodrug.

One embodiment provides a nucleic acid prodrug that has the following structure:

<img file="MX342945B_D0009.tif" />

where Rl is -OH, -SH, -NRdRd, -N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, aryl-Yl-, heteroaryl-Yl-, -P (0) (Re) 2,

HP (0) (Re), -ORa O -SRc;

Yl is 0, NRd, S or Se;

Ra is a blocking group;

Re is a blocking group;

each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (O) (Re) 2 or -HP (0) (Re);

each instance of Re is independently hydrogen,

IMPISg

MEXICAN INSTITUTE £ í “^you’““’^r

OF THE PROPERTY

INDUSTRIAL alkyl, aryl, alkenyl, alkynyl, alkyl-Y2-, alkenylY2-, alkynyl-Y2-, aryl-Y2- or heteroaryl-Y2-, or a cation which is Na + 1, Li + 1 or K + l;

Y2 is O, NRd OS;

each instance of R2 is independently hydrogen,

-OH, -SH, -NRdRd, -N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, arylYl-, heteroaryl-Yl-, -ORb or -SRc, where Rb is a blocking group;

each instance of Ba is independently blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase, · at least one instance of X is -OCH2CH2S-S (0) 2R10, -

<img file="MX342945B_D0010.tif" />

OR

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0011.tif" />

<img file="MX342945B_D0012.tif" />

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or

<img file="MX342945B_D0014.tif" />

R3 is hydrogen, a blocking group, a bonding moiety connected to a solid support, or a bonding moiety connected to a nucleic acid;

RIO is an alkyl group having 1 to 4 carbon atoms;

Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl;

MEXICAN INSTITUTE

OF IA PROPERTY

INDUSTRIAL

R12 is hydrogen or alkyl;

Z is S or O;

q is 0, 1 or 3;

W is 1, 2, 3, 4, 5, or 6;

R15 and R16 are independently hydrogen or methyl;

R17 is selected from alkyl, aryl, or a CH2CH = CH2;

<img file="MX342945B_D0015.tif" />

n is an integer from 1 to about 200.

Another embodiment provides a nucleic acid prodrug that has the following structure:

<img file="MX342945B_D0016.tif" />

where Rl is -OH, -SH, -NRdRd, -N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, aryl-Yl-, heteroaryl-Yl-, -P (O) (Re) 2,

HP (O) (Re), -ORa or -SRc;

<td></td><td></td><td></td><td></td><td></td><td>MPI</td>

<td></td><td></td><td></td><td></td><td>OR</td><td>, &lt;.tit! , r »mexiCanc Ot LA ΡΜ · ΡΙΕ · Α ' INDUSTRIAL</td>

<td>Yl</td><td>is</td><td> 0,</td><td>NRd, S</td><td>! o I know;</td><td></td>

<td>Ra</td><td>is</td><td>a</td><td>group</td><td>blocking;</td><td></td>

<td>Rc</td><td>is</td><td>a</td><td>group</td><td>blocking;</td><td></td>

<img file="MX342945B_D0017.tif" />

each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (0) (Re) 2 or -HP (O) (Re);

each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2-, alkenylY2-, alkynyl-Y2-, aryl-Y2- or heteroaryl-Y2-, or a cation that is Na + 1, Li + 1 or K + l;

<td>Y2 is</td><td>0, NRd or S</td><td> ¡;</td><td></td>

<td>every</td><td>instance</td><td>from R2</td><td>is independently hydrogen,</td>

<td>-OH, -SH,</td><td>-NRdRd,</td><td>-N3,</td><td>halogen, alkyl, alkenyl,</td>

<td>alkynyl,</td><td>alkyl-Yl</td><td colspan="2">-, alkenyl-Yl-, alkynyl-Yl-, aryl-</td>

<td colspan="2">Yl-, heteroaryl-Yl-,</td><td>-ORb</td><td>or -SRc, where Rb is a group</td>

<td>blocking;</td><td></td><td></td><td></td>

<td>every</td><td>instance</td><td>from Ba</td><td>is independently adenine,</td>

blocked or unblocked cytosine, guanine, thymine, uracil, or modified nucleobase;

<img file="MX342945B_D0018.tif" />

OR

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<img file="MX342945B_D0020.tif" />

OR

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&gt; ^I

I

<img file="MX342945B_D0022.tif" />

R3 is hydrogen, a blocking group, a bonding moiety connected to a solid support, or a bonding moiety connected to a nucleic acid;

RIO is an alkyl group having 1 to 4 carbon atoms;

Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl;

R12 is hydrogen or alkyl; and n is an integer from 1 to about 200.

IMPI

MEXICAN INSTITUTE • t industxial currency

A further embodiment provides the nucleic acid prodrug where each X-phosphonate residue of the compound of Formula 1 is more than 98% diastereomerically pure as determined by 31P NMR or reverse phase HPLC spectroscopy.

A further embodiment provides the nucleic acid prodrug where each X-phosphonate moiety has an RP configuration.

A further embodiment provides the nucleic acid prodrug where each X-phosphonate moiety has an SP configuration. A further embodiment provides the nucleic acid prodrug where each X-phosphonate independently has an RP configuration or a configuration

SP.

A further embodiment provides the nucleic acid prodrug where RIO is methyl. A further embodiment provides the nucleic acid prodrug where Rll is methyl. A further embodiment provides the nucleic acid prodrug where R12 is methyl.

A further embodiment provides the nucleic acid prodrug, where at least 25% of the X residues of the nucleic acid prodrug are independently selected

<img file="MX342945B_D0023.tif" />

ΙΜΡΙ

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

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Ar-S

F? N

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© xMe l \ k

I me me

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S ^ / x © .Me ó^I

I &lt;!)

Nx

I

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A further embodiment provides the nucleic acid prodrug, where at least 50% of the X residues of the nucleic acid prodrug are independently selected

<img file="MX342945B_D0030.tif" />

OR

OR

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MEXICAN INSTmANO

OF INDUSTRIAL PROPERTY

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_Rii

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<img file="MX342945B_D0034.tif" />

N

I

A further embodiment provides the nucleic acid prodrug, where at least 90% of the X residues of the nucleic acid prodrug are independently selected

<img file="MX342945B_D0035.tif" />

OR

<img file="MX342945B_D0036.tif" />

A further embodiment provides the nucleic acid prodrug, where each X residue of the acid prodrug

<img file="MX342945B_D0037.tif" />

nucleic is independently selected from

<img file="MX342945B_D0038.tif" />

OR

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or

S ^ x \ © .Me ¿ ^II

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I

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Y ° ^ V

NMe

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I

N

I

IMPi ΐΝΥΠΤιιτη _________

AND

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© xMe »Me

I

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A further embodiment provides the nucleic acid prodrug, where each X residue of the nucleic acid prodrug is independently selected from -OCH2CH2S-OCH2CH2S-SCH2CH2OH, -OCH2CH2CO2H,

S (O) 2R1Q,

<img file="MX342945B_D0048.tif" />

R12 O

YsAAr ,, f

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Α_ο^ ° γ^ρ or

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IMPI

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/

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Λ,

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R-ιο

<img file="MX342945B_D0058.tif" />

<img file="MX342945B_D0059.tif" />

<img file="MX342945B_D0060.tif" />

<img file="MX342945B_D0061.tif" />

<img file="MX342945B_D0062.tif" />

R 15,^R16 OR

Χ^ζΐΓ⁵Τ ^what or '17

R-is laugh v²^ -sV&gt;¹⁸ w

I jM. PI

MEXICAN INSTITUTE

FROM THE SBOPHDAD ·,

<img file="MX342945B_D0063.tif" />

R3 is hydrogen, a blocking group, a ^ this bond connected to a solid support or a moiety of "" bond connected to a nucleic acid;

RIO is an alkyl group having 1 to 4 carbon atoms;

Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl;

R12 is hydrogen or alkyl;

<td></td><td>z</td><td>is</td><td>S</td><td>u 0;</td>

<td> 10</td><td>what</td><td>is</td><td> 0,</td><td>1st</td>

<td></td><td>w</td><td>is</td><td> 1,</td><td> 2,</td>

R15 and R16 are independently hydrogen or methyl;

R17 is selected from alkyl, aryl, or a CH2CH = CH2; and

R18 is selected from N (CH3) 2,

N.

-N.

<img file="MX342945B_D0064.tif" />

-N.

| -N. NMe, and IVIeOjC

One embodiment provides a nucleic acid prodrug that has the following structure:

<img file="MX342945B_D0065.tif" />

n ₁₅ IMPI * a&gt; 5

MEXICAN INSTITUTE

FROM THE ΓΚΟΓΙΕΠΛΙ&gt; ^ 2Τ ^ ~ ΜίΓί &lt;ΐί

1N DUST IA_kL where Rl is -OH, -SH, -NRdRd, -N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, aryl-Yl-, heteroaryl-Yl- , -P (0) (Re) 2,

HP (O) (Re), -ORa or -SRc;

<td> 5</td><td>Y1 is 0, NRd, S or Se;</td>

<td></td><td>Ra is a blocking group;</td>

<td></td><td>Re is a blocking group;</td>

each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (0) (Re) 2 or -HP (O) (Re);

each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2-, alkenylY2-, alkynyl-Y2-, aryl-Y2- or heteroaryl-Y2-, or a cation that is Na + 1, Li + 1 or K + l;

Y2 is O, NRd or S;

each instance of R2 is independently hydrogen,

-OH, -SH, -NRdRd, -N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, arylYl-, heteroaryl-Yl-, -ORb or -SRc, where Rb is a blocking group;

each instance of Ba is independently blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;

IMPI

R12 O

ΛΛΛ

MEXICAN INSTITUTE&gt; s OF PROPERTY

α._

<img file="MX342945B_D0066.tif" />

at least one X is,

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&lt;Me&gt;

V^S^^ f^N'I

O; where RIO is an alkyl group having

R3 is hydrogen, a blocking group, a bonding moiety connected to a solid support, or a bonding moiety connected to a nucleic acid; and n is an integer from 1 to about 200.

A further embodiment provides a nucleic acid prodrug where RIO is methyl. A further embodiment provides a nucleic acid prodrug where Rll is methyl. A further embodiment provides a nucleic acid prodrug where R12 is methyl.

A further embodiment provides a nucleic acid prodrug, where at least 2 5% of the X residues of the

IMPI 'NSTíTlJTO MEXICANO DF IA PROPERTY

<img file="MX342945B_D0068.tif" />

nucleic acid prodrug is selected independently

<img file="MX342945B_D0069.tif" />

where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl. A further embodiment provides a nucleic acid prodrug, where at least 50% of the X residues of the nucleic acid prodrug are selected

CF

<img file="MX342945B_D0070.tif" />

Independently of

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IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0072.tif" />

<img file="MX342945B_D0073.tif" />

or

AND^S\ x-^z^ YR₁₁

O '^ nvfe

Me (me) me ^N'Me v

<img file="MX342945B_D0074.tif" />

C¡) ^ Me NMe; where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and

R12 is hydrogen or alkyl. A further embodiment provides a nucleic acid prodrug, where at least 90% of the X residues of the nucleic acid prodrug are

R12 O a_sto_ora independently selects 5 O R- | i

<img file="MX342945B_D0075.tif" />

Or me ; where RIO is a group

IMPI

MEXICAN INSTITUTE OF. IA PRONEOAP NOIJSTRIAL

<img file="MX342945B_D0076.tif" />

alkyl having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl. A further embodiment provides a nucleic acid prodrug, where each X residue of the nucleic acid prodrug is independently selected from

Ri? Or v

TO_cTO₀X_r \

<img file="MX342945B_D0077.tif" />

(j) I

-Sx / Nx.

former

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_Rii yS ^ © .Me ^X Ϊ. I^I cp me \ y⁸- ~ andⁿ-m.

O, and O where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl.

One embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the following structure:

<img file="MX342945B_D0079.tif" />

ΙΜΡΙ

MEXICAN INSTITUTE F OF PROPERTY ^λ

INDUSTRIAL where Rl is -OH, -SH, -NRdRd, -N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, aryl-Yl-, heteroaryl-Yl- , -P (O) (Re) 2,

HP (O) (Re), -ORa or -SRc;

<td> 5</td><td>Yl</td><td>is</td><td> 0,</td><td>NRd, SO Se;</td>

<td></td><td>Ra</td><td>is</td><td>a</td><td>blocking group;</td>

<td></td><td>Rc</td><td>is</td><td>a</td><td>blocking group;</td>

each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (O) (Re) 2 or -HP (O) (Re);

each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2-, alkenylY2-, alkynyl-Y2-, aryl-Y2- or heteroaryl-Y2-, or a cation that is Na + 1, Li + 1 or K + l;

Y2 is 0, NRd or S;

each instance of R2 is independently hydrogen,

-OH, -SH, -NRdRd, -N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, arylYl-, heteroaryl-Yl-, -ORb or -SRc, where Rb is a blocking group;

each instance of Ba is independently blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;

where at least one residue of X from the acid prodrug

<img file="MX342945B_D0080.tif" />

<img file="MX342945B_D0081.tif" />

<img file="MX342945B_D0082.tif" />

&lt;!)

N.

Me (¡) I am alkyl group having alkyl, aryl, heteroaryl

R12 is hydrogen or alkyl;

?

^N'I

O; where RIO is one to 4 carbon atoms; Rll is heterocyclyl or cycloalkyl; and

R3 is hydrogen, a blocking group, a residue of

ΙΜΡί ^&gt;

INSTITUTO MEXICANO oe la montr&gt; AD.

bond connected to a solid support or a moiety of '^ ftlac connected to a nucleic acid; and n is an integer from 1 to about 200;

where the method used to synthesize the nucleic acid prodrug comprises the following steps: (1) reacting a molecule comprising an achiral H-phosphonate residue and a nucleoside comprising a 5'-0H residue to form a condensed intermediate; and (2) converting the condensed intermediate to the nucleic acid prodrug comprising a chiral X-phosphonate moiety.

Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the structure of Formula 1, where each X-phosphonate residue of the compound of Formula 1 is more than 98% diastereomerically pure as determined by 31P NMR or HPLC spectroscopy. reverse phase. Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the structure of Formula 1 where each Xphosphonate moiety has an RP configuration. Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the structure of

Formula 1 where each X-phosphonate residue has an SP configuration. Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug.

IMPI

INSTITUTO MiXICANO DE LA PROilEDAI&gt;

_ industrial that has the structure of Formula 1 where each X-phosphonate independently has an RP configuration or an SP configuration.

Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the structure of Formula 1, where at least 2-5% of the X residues of the nucleic acid prodrug are independently selected from

<img file="MX342945B_D0083.tif" />

V^S^^ and^N'I

IMPI

MEXICAN INSTITUTE

DE LA PROPIEDAD '' where RIO is an alkyl group that has from 1 to^ND^^STcftom?

carbon; Rll is alkyl, aryl, heteroaryl, heterocTcTTTO or cycloalkyl; and R12 is hydrogen or alkyl. Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the structure of the

Formula 1, where at least 50% of the X residues of the nucleic acid prodrug is independently selected from ^R12 OR

Λ₀

V^S^ Y ° '^R1O V ° ^ Y ° 'R1O / oo

S 'R11 /

<img file="MX342945B_D0084.tif" />

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VR11 Ru

I ^x J. Me

S ^^ © Ale

I

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Me (me) me

AND^S'-^/Yf^xN'I

O (p ^ Me (p Me

I

Y ° —γδχζγΝ.

IMPI

INSTITUTO MEXICANO OE LA PROPIEDAD where RIO is an alkyl group that has 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl

<img file="MX342945B_D0087.tif" />

or cycloalkyl; and R12 is hydrogen or alkyl. Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the structure of the

Formula 1, where at least 90% of the X residues of the nucleic acid prodrug is independently selected from

R- | 2 O

AAA

Az '11

<img file="MX342945B_D0088.tif" />

‘10

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<img file="MX342945B_D0090.tif" />

V¹¹ v

OR

I

I

Cj) Me (pMe ySx ^ © .Me \ - &lt;Κ / Ύ^ν'_Μθ γ^δ'^ γ^Ν'Μβ ^and. oo

Me (p ^ me

V ° ^ Y^N^ e (¡) Me

AND^s^ Y%_and₂₆ IMPI ^ o INSTITUTO MEXICANO iT

OF PROPERTY V

INDUSTRIAL where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl. Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the structure of the

Formula 1, where each instance of X is selected independently of ^R12

AA,

<img file="MX342945B_D0091.tif" />

V^S'A °'^R”Or

<img file="MX342945B_D0092.tif" />

<img file="MX342945B_D0093.tif" />

AND^Rl1 Not ^I

Me, &lt;f&gt; Me -ΓΨ (f) Me

V^Sx^ ü: V ° ^ A^N'Me and^S-V Me o, o

I &lt;!)

V ° ^^^N^ Me, V ^Λ ; where RIO is

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, ariio, heteroaryl, heterocyclyl or cycloalkyl; and R12 is hydrogen or alkyl.

Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the structure of Formula 1 where RIO is methyl. Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the structure of

Formula 1 where Rll is methyl. Another embodiment provides a pharmaceutical composition comprising a nucleic acid prodrug having the structure of Formula 1 where R12 is methyl.

One embodiment provides a method of treating an upregulated RNase L-associated disease by administering a therapeutic amount of a chiral nucleic acid prodrug. Another modality provides a method of treating a disease associated with

RNase L up-regulated, where the disease is chronic fatigue syndrome. Another embodiment provides a method of treating a down-regulated RNase L-associated disease by administering a therapeutic amount of a chiral nucleic acid prodrug. Another modality provides a method of treating a down-regulated RNase L-associated disease, where the

<img file="MX342945B_D0094.tif" />

disease is cancer. In another modality, the cancer is

IMPI

MEXICAN INSTITUTE

OWNERSHIP / * -

INDUSTRIAL selects from prostate, colorectal and pancreatic cancer.

In one embodiment, the downregulated RNase L cancer is pancreatic cancer. In another embodiment, the cancer with downregulated RNase L is prostate cancer. In yet another embodiment, the cancer with downregulated RNase L is colorectal cancer.

One embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a nucleic acid prodrug having the following structure:¹

R²

Ba

R²

X&gt;_px '—I ίΊ θ *

V^or and

OR³Formula 1 where Rl is -OH, -SH, -NRdRd, -N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, aryl-Yl-, heteroaryl-Yl -,

P (O) (Re) 2, -HP (O) (Re), -ORa or -SRc;

<td>Y1</td><td>is</td><td> 0,</td><td>NRd,,</td><td>S or Se;</td>

<td>Ra</td><td>is</td><td>a</td><td>group</td><td>blocking;</td>

<td>Re</td><td>is</td><td>a</td><td>group</td><td>blocking;</td>

each instance of Rd is independently hydrogen,

<img file="MX342945B_D0095.tif" />

alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (O) (Re) 2 or -HP (O) (Re);

each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2-, alkenyl5 Y2-, alkynyl-Y2-, aryl-Y2-, or heteroaryl-Y2-, or a cation that is Na + 1, Li +1 or K + l;

Y2 is 0, NRd or S;

each instance of R2 is independently hydrogen, OH, -SH, -NRdRd, -N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, arylYl-, heteroaryl-Yl-, -ORb or -SRc, where Rb is a blocking group;

each instance of Ba is independently blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;

at least one X residue of the nucleic acid prodrug is independently selected from

<img file="MX342945B_D0096.tif" />

O o

TMPI '* ^Γ”ΤΠ MEXICAN 'PROPERTY •' STRtAL

<img file="MX342945B_D0097.tif" />

<img file="MX342945B_D0098.tif" />

z _Rii

OR

<img file="MX342945B_D0099.tif" />

or _Rl) ¿ ^IMe © xMe&gt; e

I

<img file="MX342945B_D0100.tif" />

&lt;!) ^Nm

I

<img file="MX342945B_D0101.tif" />

I

V ° ^ Y^N'^I V^S^ y^N'Me o, and O where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl;

R3 is hydrogen, a blocking group, a bonding moiety connected to a solid support, or a bonding moiety connected to a nucleic acid; and n is an integer from 1 to about 200;

0 RIO is an alkyl group having 1 to 4 carbon atoms;

where the method used to synthesize the nucleic acid prodrug comprises the following steps: (1) reacting a molecule comprising an H-phosphonate moiety

<img file="MX342945B_D0102.tif" />

achiral and a nucleoside comprising a 5'-OH moiety to form a condensed intermediate; and (2) converting the condensed intermediate to the nucleic acid prodrug comprising a chiral X-phosphonate moiety.

Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound of Formula 1, where at least 25% of the X residues of the nucleic acid prodrug are independently selected from

<img file="MX342945B_D0103.tif" />

or

X

X.

γ ° &lt;j&gt; 'Me Nh

I

IMPI

-MEXICAN TITLE ~^r '.A ΓΚΟΜΕΡΛΡ To

PIKTRIAL φ Me N.

Me where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl. Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound of

Formula 1, where at least 50% of the X residues of the nucleic acid prodrug is independently selected from. R12 O R12 Q &lt;

or

Rl2 O

AAA „„ ^Λ

<img file="MX342945B_D0104.tif" />

^k10

So ^^s'r

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\ ζ

O ^ XMe ' ^IMe \ '(pMí φΜ &lt;

VS¡¡ »V ° ^ Y^N^ - ^s^N

I ,

I

<img file="MX342945B_D0106.tif" />

ungodly

MIXICAN INSTITUTE (&lt;-. I heard THE OWN »

JOUSTXIAL V

I

O me

Y ° ^ Y% e O where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl. Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound of

Formula 1, where at least 90% of the X residues of the nucleic acid prodrug is independently selected from

<img file="MX342945B_D0107.tif" />

'.'I

Me (j) me ^N'_I

<img file="MX342945B_D0108.tif" />

<img file="MX342945B_D0109.tif" />

¡&lt;¡&gt; Me o. and

<img file="MX342945B_D0110.tif" />

X Men.

I

IMPL · · ¡* '-πτυτο MEXICAN ¿2 · **' ·

OsTMAL where RlO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl. Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound of

Formula 1, where each instance of X is selected independently of

R12 9 \ / O.

/\_QÁ „Á_D

SO Riί o ^ 10

<img file="MX342945B_D0111.tif" />

'R ° \ ^ Ri ^ Ru

O, O and_{Rl1 R11} yS ^ © .Me ¿¿. I^I &lt;¡) Me N

I

O Me \ ^I V ^ T '^

IMPI

MEXICAN INSTITUTE where RIO is an alkyl group that has from 1 to 4

Dt LA FROFIEOAl i INDUSTRIAL

<img file="MX342945B_D0112.tif" />

carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl.

Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound of Formula 1, where RIO is methyl. Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound of Formula 1, where Rll is methyl. Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound of

Formula 1, where R12 is methyl.

Another embodiment provides a method of treating cancer which comprises administering a therapeutic amount of a compound of Formula 1, where each X-phosphonate residue of the compound of Formula 1 is more than 98% diastereomerically pure as determined by 31P NMR spectroscopy or Reverse phase HPLC. Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound of Formula 1, where each residue of

X-phosphonate has an RP configuration. Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound of

Formula 1, where each X-phosphonate residue has a

IMPIs

MEXICAN INSTITUTE

6 OF THE PROPERTY

INDUSTRIAL configuration SP. Another embodiment provides a method of treating cancer comprising administering a therapeutic amount of a compound of Formula 1, where each Xphosphonate independently has either an RP configuration or an SP configuration.

Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound of Formula 1, where the cancer is pancreatic cancer.

One embodiment provides a nucleic acid prodrug that has the following structure:

<img file="MX342945B_D0113.tif" />

where Rl is -OH, -SH, -NRdRd, -N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, aryl-Yl-, heteroaryl-Yl-, -P (O) (Re) 2,

HP (0) (Re), -ORa or -SRc;

Yl is 0, NRd, S or Se;

Ra is a blocking group;

Rc is a blocking group;

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0114.tif" />

each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (0) (Re) 2 or -HP (O) (Rejected instance of Re is independently hydrogen, alkyl, aryl , alkenyl, alkynyl, alkyl-Y2-, alkenylY2-, alkynyl-Y2-, aryl-Y2- or heteroaryl-Y2-, or a cation which is Na + 1, Li + 1 or K + 1;

<td colspan="3">Y2 is 0, NRd OS;</td><td colspan="2" rowspan="2">is independently hydrogen,</td>

<td>every</td><td>instance of</td><td>R2</td>

<td>-OH, -SH,</td><td>-NRdRd,</td><td>N3,</td><td>halogen,</td><td>alkyl, alkenyl,</td>

<td>alkynyl,</td><td>alkyl-Yl-,</td><td colspan="2">alkenyl-Yl-,</td><td>alkynyl-Yl-, aryl-</td>

Yl-, heteroaryl-Yl-, -ORb or -SRc, where Rb is a blocking group;

each instance of Ba is independently blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;

at least one instance of X is

<img file="MX342945B_D0115.tif" />

OR

OR

<img file="MX342945B_D0116.tif" />

Υ ° ^ γ% θ Υ^δ''^/'γ^Ν'Μθ O, O o

R3 is hydrogen, a blocking group, a bonding moiety connected to a solid support, or a bonding moiety connected to a nucleic acid;

RIO is an alkyl group having 1 to 4 carbon atoms;

Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl;

R12 is hydrogen or alkyl; and n is an integer from 1 to about 200.

One embodiment provides a nucleic acid prodrug of Formula 2, where each X-phosphonate residue of the compound of Formula 2 is more than 98% diastereomerically pure as determined by 31P NMR or reverse phase HPLC spectroscopy. Another modality provides a prodrug of₃₉ IMPIr&gt;

^-³ MEXICAN INSTITUTE rl · · '·? *'¹

OF THE PROPERTY

INDUSTRIAL ·.

Nucleic acid of Formula 2, where each X-phosphonate residue has an RP configuration. Another embodiment provides a nucleic acid prodrug of Formula 2, where each X-phosphonate residue has an SP configuration. Another embodiment provides a nucleic acid prodrug of Formula 2, where each X-phosphonate has either an RP configuration or an SP configuration.

Another embodiment provides a nucleic acid prodrug of Formula 2 where RIO is methyl. Another embodiment provides a nucleic acid prodrug of Formula 2 where Rll is methyl. Another embodiment provides a nucleic acid prodrug of Formula 2 where R12 is methyl.

Another embodiment provides a nucleic acid prodrug of Formula 2, where at least 25% of the X residues of the nucleic acid prodrug are selected independently of

<img file="MX342945B_D0117.tif" />

© .Me

I

<img file="MX342945B_D0118.tif" />

(pMe

I

IMPI

INSTITUTO mzxican; SAY THE INDUSTRIAL PKOFIEDAr.

<img file="MX342945B_D0119.tif" />

Y ^ TSle ⁵ φ Me &lt;¡&gt; Me γ^ο-γ% ® V^Sx^ V^N'^I

Or me

Another embodiment provides a nucleic acid prodrug of Formula 2, where at least 50% of the X residues of the nucleic acid prodrug are selected independently of

<img file="MX342945B_D0120.tif" />

O o

() I

Νχ

I

V^S^ And o

IMPI

<img file="MX342945B_D0121.tif" />

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

Another embodiment provides a nucleic acid prodrug of Formula 2, where at least 90% of the X residues of the nucleic acid prodrug are selected independently of

<img file="MX342945B_D0122.tif" />

or

IMPI

<img file="MX342945B_D0123.tif" />

INSTITUTO MtX ICA NO OF. INDUSTRIAL PROPERTY

Another embodiment provides an aci nucleic prodrug of Formula 2, where each residue of X in the nucleic acid prodrug is independently selected from ^ 12 Q Rl2 0

Ad ^ O ^ R -, -, / ^ S ^ O ^ Rn \^SY ° 'P.

AND^s—Γ%₁₀ γ ° ^% „/ __s or O ^{z Q}

<img file="MX342945B_D0124.tif" />

© .Me ^ Me

Me (j) me

Y ° ^ Y^N'I

V ”or

© .Me Me (j) Me

V^s--AND^N^^I

OR

\ .O &lt;p ^ Me M (¡f ^ Me

V^s^ V^N^^I or, and θ

One embodiment provides a pharmaceutical composition.

I understand that it comprises a nucleic acid prodrug that has the following structure:

<img file="MX342945B_D0125.tif" />

Formula 2

IMPI Mexican institute OF PROPERTY where Rl is -OH, -SH, -NRdRd, -N3, halogen

<img file="MX342945B_D0126.tif" />

alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl "and alkynyl-Yl-, aryl-Yl-, heteroaryl-Yl-, -P (0) (Re) 2,

HP (O) (Re), -ORa or -SRc;

<td> 5</td><td>Yl</td><td>is</td><td> 0,</td><td>NRd,</td><td>S or Se;</td>

<td></td><td>Ra</td><td>is</td><td>a</td><td>group</td><td>blocking;</td>

<td></td><td>Re</td><td>is</td><td>a</td><td>group</td><td>blocking;</td>

each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (0) (Re) 2 or -HP (O) (Re);

each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2-, alkenylY2-, alkynyl-Y2-, aryl-Y2- or heteroaryl-Y2-, or a cation that is Na + 1, Li + 1 or K + l;

Y2 is 0, NRd or S;

each instance of R2 is independently hydrogen,

-OH, -SH, -NRdRd, -N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, arylYl-, heteroaryl-Yl-, -QRb or -SRc, where Rb is a blocking group;

each instance of Ba is independently blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;

where at least one residue of X from the acid prodrug

Nucleic IMPI is selected independently of

<img file="MX342945B_D0127.tif" />

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0128.tif" />

where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl;

R3 is hydrogen, a blocking group, a bonding moiety connected to a solid support, or a bonding moiety connected to a nucleic acid; and

IMPI

INSTITUTO MEXtCANO DF. INDUSTRIAL PROPERTY

<img file="MX342945B_D0129.tif" />

, IPL'U.'IMnL - - 1n is an integer from 1 to about 200;

where the method used to synthesize the nucleic acid prodrug comprises the following steps: (1) reacting a molecule comprising an achiral H-phosphonate residue and a nucleoside comprising a 5'-OH residue to form a condensed intermediate; and (2) converting the condensed intermediate to the nucleic acid prodrug comprising a chiral X-phosphonate moiety.

Another embodiment provides a pharmaceutical composition comprising a compound of Formula 2, where each residue of

X-phosphonate of the compound of Formula 2 is more than 98% diastereomerically pure as determined by 31P NMR or reverse phase HPLC spectroscopy. Another embodiment provides a pharmaceutical composition comprising a compound of Formula 2, where each X-phosphonate moiety has an RP configuration. Another embodiment provides a pharmaceutical composition comprising a compound of

Formula 2, where each X-phosphonate moiety has an SP configuration. Another embodiment provides a pharmaceutical composition comprising a compound of Formula 2, where each X-phosphonate independently has either an RP configuration or an SP configuration.

Another embodiment provides a pharmaceutical composition comprising a compound of Formula 2, where at least the

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0130.tif" />

25% of the X residues of the nucleic acid prodrug are selected independently of

<img file="MX342945B_D0131.tif" />

where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl. Another embodiment provides a pharmaceutical composition comprising a

ΙΜΡΓ $ |

Λ 7 MEXICAN INSTITUTE 'PE LA MONEOaP Ontei

INDUSTRIAL composed of Formula 2, where at least 50% of the X residues of the nucleic acid prodrug is independently selected from

<img file="MX342945B_D0132.tif" />

<img file="MX342945B_D0133.tif" />

γ^δ^ ν ^ χθγ_Ρι1

<img file="MX342945B_D0134.tif" />

© .Me

Nx • Me Me

© .Me

I

<img file="MX342945B_D0135.tif" />

<img file="MX342945B_D0136.tif" />

<img file="MX342945B_D0137.tif" />

I

Me where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl. Another modality

IMPI

MEXICAN INSTITUTE PE INDUSTRIAL PROPERTY

<img file="MX342945B_D0138.tif" />

provides a pharmaceutical composition comprising a compound of Formula 2, where at least 90% of the X residues of the nucleic acid prodrug are independently selected from

<img file="MX342945B_D0139.tif" />

<img file="MX342945B_D0140.tif" />

where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl. Another modality

IMPI »:

MEXICAN INSTITUTE

OE &lt;-INDUSTRIAL PROPERTY provides a pharmaceutical composition comprising a compound of Formula 2, where each instance of X is independently selected from

<img file="MX342945B_D0141.tif" />

<img file="MX342945B_D0142.tif" />

or, and O where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl.

Another embodiment provides a pharmaceutical composition comprising a compound of Formula 2 where RIO is methyl.

Another embodiment provides a pharmaceutical composition that

0 MEXICAN INSTITUTE

OF THE PROPERTY ^&lt;^ j ~~ «W^í4íitEy

INDUSTRIAL -— comprises a compound of Formula 2 where Rll is methyl. Other

ΙΜΡΙ embodiment provides a pharmaceutical composition comprising a compound of Formula 2 where R12 is methyl.

One embodiment provides a method of treating cancer, which comprises administering a therapeutic amount of a nucleic acid prodrug having the following structure:

<img file="MX342945B_D0143.tif" />

where Rl is -OH, -SH, -NRdRd, -N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, 15 alkynyl-Yl-, aryl-Yl-, heteroaryl-Yl- , -P (0) (Re) 2,

HP (O) (Re), -ORa or -SRC;

<td>Y1</td><td>is</td><td> 0,</td><td>NRd,,</td><td>S or Se;</td>

<td>Ra</td><td>is</td><td>a</td><td>group</td><td>blocking;</td>

<td>Re</td><td>is</td><td>a</td><td>group</td><td>blocking;</td>

each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (0) (Re) 2 or -HP (O) (Re);

each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2-, alkenyl51

ΙΜΡΙ

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0144.tif" />

Y2-, alkynyl-Y2-, aryl-Y2- or heteroaryl-Y2-, or a cation that is Na + 1, Li + 1 O K + l;

Y2 is O, NRd OS;

each instance of R2 is independently hydrogen,

-OH, -SH, -NRdRd, -N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, arylYl-, heteroaryl-Yl-, -ORb or -SRc, where Rb is a blocking group;

each instance of Ba is independently blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;

at least one X residue of the nucleic acid prodrug is independently selected from

<img file="MX342945B_D0145.tif" />

<img file="MX342945B_D0146.tif" />

\'

R „• Oxz \ © .Me

Nx

Λ ^I

I

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V

IMPI

Ν '

I

Me (pMe N.

I

Y ° ^%_and or

TO

O me

Υ ° ^^ γ^Ν'Μ® Y

<img file="MX342945B_D0148.tif" />

I

Y ► Me where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl;

R3 is hydrogen, a blocking group, a bonding moiety connected to a solid support, or a bonding moiety connected to a nucleic acid; and n is an integer from 1 to about 200;

RIO is an alkyl group having 1 to 4 carbon atoms;

where the method used to synthesize the nucleic acid prodrug comprises the following steps: (1) reacting a molecule comprising an achiral H-phosphonate residue and a nucleoside comprising a 5'-OH residue to form a condensed intermediate; and (2) converting the condensed intermediate to the nucleic acid prodrug comprising a chiral X-phosphonate moiety.

IMPI

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

Another embodiment provides a method of treating cancer, which comprises administering a therapeutic amount of a compound having the structure of Formula 2, where at least 25% of the X residues of the nucleic acid prodrug are independently selected from

<img file="MX342945B_D0149.tif" />

<img file="MX342945B_D0150.tif" />

<img file="MX342945B_D0151.tif" />

&lt;? ^II

J? Me

I

I &lt;!)

AND^s'^ Y^NMe

Am) I

I ?

(p Me \^S^ Yf^N'Me where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl

ΙΜΡΙ

INSTITUTO MEXICANO OE LA PXONEDAO INDUSTRIAL or cycloalkyl; and R12 is hydrogen or alkyl. Another modality

<img file="MX342945B_D0152.tif" />

provides a method of treating cancer, which comprises administering a therapeutic amount of a compound having the structure of Formula 2, where at least 50% of the X residues of the nucleic acid prodrug are independently selected from

<img file="MX342945B_D0153.tif" />

<img file="MX342945B_D0154.tif" />

O ^ / \ © .Me&gt;? Me

I

OR

<img file="MX342945B_D0155.tif" />

OR

OR

IMPI

INSTTTUTT 'MEXICANO

INSTO V_{LA nope},_Fda^ .

where RIO is an alkyl group having 1 atoms of carbonQ; Rll is alkyl, aryl, heteroaryl, ~ heterocyclyl or cycloalkyl; and R12 is hydrogen or alkyl. Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound having the structure of Formula 2, where at least 90% of the X residues of the nucleic acid prodrug are independently selected from

<img file="MX342945B_D0156.tif" />

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0157.tif" />

where RIO is an alkyl group having 1 to 4 atoms

........... T of carbon; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl. Another embodiment provides a method of treating cancer, which comprises administering a therapeutic amount of a compound having the structure of Formula 2, where each instance of X is independently selected from

<img file="MX342945B_D0158.tif" />

and_{Rll R] 1} o o me v^s-r &lt;? me

N.

Me where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl.

Another embodiment provides a method of treating cancer which comprises administering a therapeutic amount of

IMPI

INSTITUTO MEXICANO DE IA FROFIEDAD INDUSTRIAL a compound that has the structure of Formula 2, where RIO is methyl. Another embodiment provides a method of treating cancer comprising administering a therapeutic amount of a compound having the structure of Formula 2, where

Rll is methyl. Another embodiment provides a method of treating cancer which comprises administering a therapeutic amount of a compound having the structure of

Formula 2, where R12 is methyl.

Another embodiment provides a method of treating cancer, which comprises administering a therapeutic amount of a compound having the structure of Formula 2, where each X-phosphonate residue of the compound of Formula 2 is more than 98% diastereomerically pure as determined by the 31P NMR spectroscopy or reverse phase HPLC.

Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound having the structure of Formula 2, where each X-phosphonate moiety has an RP configuration. Another embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound having the structure of Formula 2, where each X-phosphonate moiety has an SP configuration. Another embodiment provides a method of treating cancer, which comprises administering a therapeutic amount of a

<img file="MX342945B_D0159.tif" />

IMPI 'NSTITUTO MEXICANO

OF THE 'NPUSTRIAL PROPERTY

<img file="MX342945B_D0160.tif" />

compound having the structure of Formula 2, where each Xphosphonate independently has an RP configuration or an SP configuration.

One embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound having the structure of Formula 2, where the cancer is pancreatic cancer.

One embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound having the structure of Formula 2, where the compound has the following formula:

<img file="MX342945B_D0161.tif" />

Formula A3-2 where each A is adenine and each Rll is independently selected from alkyl, aryl, heteroaryl, heterocyclyl, and cycloalkyl. A further embodiment provides a method of treating pancreatic cancer comprising administering a therapeutic amount of a compound of Formula A3-2.

IMPI ^

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

One embodiment provides a method of treating cancer that comprises administering a therapeutic amount of a compound having the structure of Formula 2, where the compound has the following formula:

<img file="MX342945B_D0162.tif" />

Formula A3-3.

A further embodiment provides a method of treating pancreatic cancer comprising administering a therapeutic amount of a compound of Formula A3-3.

One embodiment provides a compound or its pharmaceutically acceptable salt having the following formula:

<img file="MX342945B_D0163.tif" />

HO OH

Formula A3-1 where each A is adenine; and at least

<img file="MX342945B_D0164.tif" />

r \ i2

ΛΑΛ a

<img file="MX342945B_D0165.tif" />

IMPI

MEXICAN INSTITUTE OF LA MONEDAD rest Wes

<img file="MX342945B_D0166.tif" />

<img file="MX342945B_D0167.tif" />

O, OO where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl.

Another embodiment provides a compound or its pharmaceutically acceptable salt having the structure of

Formula A3-1, where at least two of the X residues of the nucleic acid prodrug are independently

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0168.tif" />

select

<img file="MX342945B_D0169.tif" />

\°'_r^NMe v^S^ Y^N'I

O, and O where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl.

Another embodiment provides a compound or its pharmaceutically acceptable salt having the structure of

Formula A3-1, where at least three of the X residues of the

IMPI

INSTITUTO MEXICANO Dk LA PROPIEDAD INDUSTRIAL

<img file="MX342945B_D0170.tif" />

nucleic acid prodrug are independently selected from

<img file="MX342945B_D0171.tif" />

oo, and O where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocycyl, or cycloalkyl; and R12 is hydrogen or alkyl.

IMPI

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL

Another embodiment provides a compound or its salt.

<img file="MX342945B_D0172.tif" />

pharmaceutically acceptable it has the structure of

Formula A3-1, where each X residue of the nucleic acid prodrug is independently selected from

<img file="MX342945B_D0173.tif" />

where RIO is an alkyl group having 1 to 4 carbon atoms; Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; and R12 is hydrogen or alkyl.

IMPI

MIXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0174.tif" />

Another embodiment provides a compound or its pharmaceutically acceptable salt having the structure of

Formula A3-1, where the compound has the following formula:

<img file="MX342945B_D0175.tif" />

INCORPORATION THROUGH THIS REFERENCE

All publications and patent applications described herein are incorporated herein in their entirety by this reference with the same scope as if each individual publication or patent application is specifically and individually indicated as incorporated herein by this reference. .

BRIEF DESCRIPTION OF THE FIGURES

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which sets forth

ΙΜΡΙ

INSTITUTO MEXICANO DE IA PROPIEDAD illustrative modalities, using the principles of the invention and the attached drawings. '

Figure 1 provides a representative analytical HPLC profile for compound 64 and GSH.

Figure 2 provides a representative HPLC profile of compound 64a, a glutathione adduct and the final product after release of the pro-residue.

Figure 3 provides a graph of conversion over time for compound 64a and 64b.

Figure 4 provides a reaction time course as determined by LC-MS for glutathione-assisted prodrug release of compound 64a.

DETAILED DESCRIPTION OF THE INVENTION

The section titles used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or parts of documents cited in the application that include, but are not limited to, patents, patent applications, articles, books, manuals and treaties are expressly incorporated herein in their entirety by this reference whatever the purpose.

Unless otherwise stated, the following terms used in the present application, which include the specification and claims, have the

<img file="MX342945B_D0176.tif" />

ΙΜΡΙ ~

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0177.tif" />

definitions provided below. It should be noted that, as used in this specification and in the appended claims, the singular forms un / una and el / la include plural referents, unless the context clearly indicates otherwise. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are employed. In this application, the use of o or y means and / or, unless otherwise indicated. Likewise, the use of the term including, as well as other forms such as including / ne included / a is not limiting.

Specific chemical terminology

Unless otherwise indicated, the use of general chemical terms, such as, but not limited to, alkyl, amine, aryl is not substituted.

As used herein, Cl-Cx includes C1-C2, C1-C3. . . Cl-Cx. By way of example only, a group called C1-C4 indicates that there are from one to four carbon atoms in the remainder, i.e. groups containing 1 carbon atom, 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms. carbon, as well as the C1-C2 and C1-C3 ranges. Accordingly, by way of example only, C1-C4 alkyl indicates that there are from one to four atoms of

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7 MEXICAN INSTITUTE

OF THE ΡΕΟΓΙΕΓΆΙ&gt; VjeBoaSw

INDUSTRIAL ”-carbon in the alkyl group, that is, the alkyl group is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and t-butyl. Whenever it appears here, a numerical range such as 1 to 10 refers to each integer in the given range, eg. , 1 to 10 carbon atoms means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, carbon atoms, 8 atoms of carbon, 9 carbon atoms or 10 carbon atoms.

The terms heteroatom or hetero, as used herein, alone or in combination, refer to an atom other than carbon or hydrogen. Heteroatoms can be independently selected from oxygen, nitrogen, sulfur, phosphorous, silicon, selenium, and tin, but are not limited to these atoms. In embodiments where two or more heteroatoms are present, the two or more heteroatoms may be the same as the rest, or some or the two or more heteroatoms may be different from the rest.

The term alkyl, as used herein, alone or in combination, refers to a straight chain or branched chain saturated hydrocarbon monoradical having from one to about ten carbon atoms or from one to six carbon atoms. Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl-l-

<img file="MX342945B_D0178.tif" />

MEXICAN INSTITUTE .u

OF PROPERTY _ industriai propyl, 2-methyl-2-propyl, 2-methyl-l-butyl, 3-methyl-lbbutyl, 2-methyl-3-butyl, 2,2-dimethyl-l-propyl, 2-methyl -pentyl, 3-methyl-l-pentyl, 4-methyl-l-pentyl, 2-methyl-2pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-lbbutyl, 3 , 3-dimethyl-l-butyl, 2-ethyl-l-butyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, tert-amyl, and hexyl, and longer alkyl groups , such as heptyl, octyl, and the like. Wherever it appears herein, a numerical range such as ClC6 alkyl or Cl-6 alkyl means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms. carbon or carbon atoms. In one embodiment, the alkyl is substituted. Unless otherwise indicated, the alkyl is unsubstituted.

The term alkenyl, as used herein, alone or in combination, refers to a straight-chain or branched-chain hydrocarbon monoradical having one or more carbon-carbon double bonds and having two to about ten carbon atoms. carbon, or two to about six carbon atoms. The group may be in the cis or trans conformation at the double bond (s), and should be understood to include both isomers. Examples include, but are not limited to, ethenyl (-CH = CH2), 1-propeni

<img file="MX342945B_D0179.tif" />

1,3 (-CH2CH = CH2), isopropenyl [-C (CH3) = CH2], butenyl butadienyl and the like. Whenever it appears here, a numerical range such as C2-C6 alkenyl or alkenyl

C2-6 means that the alkenyl group can consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, carbon atoms, or 6 carbon atoms. In one embodiment, alkenyl is substituted. Unless otherwise indicated, alkenyl is unsubstituted.

The term alkynyl, as used herein, alone or in combination, refers to a straight-chain or branched-chain hydrocarbon monoradical having one or more carbon-carbon triple bonds and having from two to about ten atoms of carbon, or two to about six carbon atoms. Examples include, but are not limited to, ethynyl, 2-propynyl, 2-butynyl, 1,3-butadiinyl, and the like. Whenever it appears herein, a numerical range such as C2C6 alkynyl or C2-6 alkynyl means that the alkynyl group may consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms. carbon.

In one embodiment, the alkynyl is substituted. Unless otherwise indicated, alkynyl is unsubstituted.

The terms heteroalkyl, heteroalkenyl

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0180.tif" />

Heteroalkynyl, as used herein, alone or in combination, refers to alkyl, alkenyl and alkynyl structures respectively, as described above, in which one or more of the skeletal chain carbon atoms (and any Associated hydrogen atom, as appropriate) are each independently replaced by a heteroatom (i.e. a non-carbon atom, such as, non-limitingly, oxygen, nitrogen, sulfur, silicon, phosphorus, tin or combinations thereof), or a heteroatomic group such as, but not limited to, -0-0-, -SS-, -0-S-, -SO-, = NN =, -N = N -, -N = N-NH-, P (0) 2-, -OP (O) 2-, -P (0) 2-0-, -S (0) -, -S (0) 2-, -SnH2- and the like.

The terms haloalkyl, haloalkenyl, and haloalkynyl, as used herein, alone or in combination, refer to alkyl, alkenyl, and alkynyl groups, respectively, as defined above, in which one or more hydrogen atoms are replaced by fluoro, chloro, bromo or iodo atoms, or combinations thereof. In some embodiments, two or more hydrogen atoms can be replaced by halogen atoms that are the same as the rest (eg. , difluoromethyl); in other embodiments two or more hydrogen atoms can be replaced by halogen atoms that are not all the same as the rest (eg 120 chloro-l-fluoro-l-iodoethyl). Non-limiting examples of

IMPI

MEXICAN INSTITUTE C4 **** U¡3 ^ »E THE PROPERTY

INDUSTRIAL haloalkyl groups are fluoromethyl, chloromethyl and bromoethyl. A non-limiting example of a haloalkenyl group is bromoethenyl. A non-limiting example of a haloalkynyl group is chloroethynyl.

The term carbon chain, as used herein, alone or in combination, refers to any alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl group, which is linear, cyclic, or any combination thereof. If the chain is part of a bond and that bond comprises one or more rings as part of a nucleus structure, for the purpose of calculating the length of the chain, the chain only includes the carbon atoms that make up the bottom or top of a certain ring and not both, and when the length of the bottom and top of the rings is not equivalent, the shortest distance will be used to determine the length of the chain. If the chain contains heteroatoms as part of the backbone, those atoms are not calculated as part of the length of the carbon chain.

The term cycloalkyl, as used herein, alone or in combination, refers to a saturated monoradical hydrocarbon ring, containing from three to about fifteen ring carbon atoms or from three

<img file="MX342945B_D0181.tif" />

to about ten ring carbon atoms, although it may include additional non-ring carbon atoms, as substituents (eg, methylcyclopropyl). Wherever present, a numerical range such as C3-C6 cycloalkyl or C3-6 cycloalkyl, means that the cycloalkyl group may consist of 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, that is, it is cyclopropyl, cyclobutyl, cyclopentyl, or cycloheptyl, although the present definition also encompasses the occurrence of the term cycloalkyl when no numerical range is indicated. The term includes fused, unfused, bridged, and spiro radicals. A fused cycloalkyl can contain two to four fused rings when the linker ring is a cycloalkyl ring and the other individual rings can be alicyclic, heterocyclic, aromatic, heteroaromatic, or any combination thereof. Examples include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, decalinyl, and bicyclo [2.2.1] heptyl and adamantyl ring systems. Illustrative examples include, but are not limited to, the following residues:

&gt;. □. or

Or and the like.

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OF PROPERTY and—,

INDUSTRIAL

In one embodiment, the cycloalkyl is substituted. Unless otherwise indicated, cycloalkyl is unsubstituted.

The terms "non-aromatic heterocyclyl" and "heteroalicyclyl", as used herein, alone or in combination, refer to saturated, partially unsaturated or fully unsaturated non-aromatic ring monoradicals containing from three to about twenty ring atoms, where one or more of the ring atoms are an atom other than carbon, independently selected from oxygen, nitrogen, sulfur, phosphorous, silicon, selenium and tin, but without limitation to these atoms. In embodiments where two or more heteroatoms are present in the ring, the two or more heteroatoms may be the same as the remainder, or some or the two or more heteroatoms may be different from the remainder. Terms include fused, unfused, bridged, and spiro radicals. A fused non-aromatic heterocyclic radical can contain two to four fused rings when the linker ring is a non-aromatic heterocycle and the other individual rings can be alicyclic, heterocyclic, aromatic, heteroaromatic, or any combination thereof. Fused ring systems can be fused by a single bond or bond

IMPI

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

<img file="MX342945B_D0182.tif" />

double, as well as through carbon-carbon, carbon-heteroatom or heteroatom-heteroatom bonds. The terms also include radicals having from three to about twelve skeletal ring atoms, as well as radicals having from three to about ten skeletal ring atoms.

The attachment of a non-aromatic heterocyclic subunit to its main molecule can be through a heteroatom or carbon atom. Also, the additional substitution can occur through a heteroatom or a carbon atom. As a non-limiting example, a non-aromatic imidazolidine heterocycle may be attached to a main molecule through its N atoms (imidazolidin-l-yl or imidazolidin-3yl) or any of its carbon atoms (imidazolidin-2yl, imidazolidin-4 -yl or imidazolidin-5-yl). In some embodiments, non-aromatic heterocycles contain one or more carbonyl or thiocarbonyl groups, such as, for example, oxo and thio containing groups. Examples include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidine, morpholino, thioxinyl, thiexyl, ethynyl, ethynyl, ethyleneyl, thienyl, tylenyl, oxethanyl ,

1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl,

IMPI ¿SlTc

MEXICAN INSTITUTE

ΠΓ I λ CDmiTn. n Z *. ** Industrial V3 indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3azabicyclo [, 3.1] hexacylanyl [3.0] 4.1.0] Heptanil, 3Hindolyl, and Quinolizinyl. Illustrative examples of heterocycloalkyl groups, also called non-aromatic heterocycles, include:

Ó O o '0' CO Λ q „hCh '° \ —ρ y - y ¿F;

or · O00uoCQ or

¿A o

NH 'HN ^ NH

Ck A.0 zS ° 'V ° r^b'NH' or '^H V 'O and the like

The terms also include all ring forms of carbohydrates, which include, but are not limited to, monosaccharides, disaccharides, and oligosaccharides. In a non-aromatic embodiment, the heterocyclyl heteroalicyclyl is substituted. Unless otherwise indicated, the non-aromatic heterocyclyl or heteroalicyclyl is unsubstituted.

The term Aryan, as used herein, alone or in combination, refers to a radical of

IMPI

INSTITUT ·,) MEXICAN ffZL · '

OF THE PROPERTY

INDUSTRIAL ^^ 5.

hi aromatic carbon six to about twenty ring carbon atoms and includes fused and unfused aryl rings. A fused aryl ring radical contains two to four fused rings when the linker ring is an aryl ring and the other individual rings can be alicyclic, heterocyclic, aromatic, heteroaromatic, or any combination thereof. Also, the term aryl includes fused and unfused rings containing from six to about twenty ring carbon atoms, as well as those containing from six to about ten ring carbon atoms. A non-limiting example of a single-ring aryl group includes phenyl; a fused ring aryl group includes naphthyl, phenanthrenyl, anthracenyl, azulenyl; and an un-fused biaryl group includes biphenyl. In one embodiment, the aryl is substituted. Unless otherwise indicated, aryl is unsubstituted.

The term heteroaryl, as used herein, alone or in combination, refers to aromatic monoradicals containing from about five to about twenty skeletal ring atoms, where one or more of the ring atoms is a heteroatom independently selected from oxygen, nitrogen, sulfur, phosphorous, silicon, selenium and tin, but not limited to these atoms and on the condition that the

IMPI

<img file="MX342945B_D0183.tif" />

ring of said group does not contain two adjacent 0 or S atoms. In embodiments where two or more heteroatoms are present in the ring, the two or more heteroatoms may be the same as the remainder, or some or the two or more heteroatoms may be different from the remainder. The term heteroaryl includes fused and unfused heteroaryl radicals having at least one heteroatom. The term heteroaryl also includes fused and unfused heteroaryls having from five to about twelve skeletal ring atoms, as well as those having from five to about ten skeletal ring atoms. Binding to a heteroaryl group can occur through a carbon atom or a heteroatom. Thus, as a non-limiting example, an imidazole group can be attached to a main molecule through any of its carbon atoms (imidazole-2-yl, imidazole-4-yl or imidazole-5-yl), or their nitrogen atoms (imidazol-l-yl or imidazol-3-yl). Also, a heteroaryl group may be further substituted by all or any of its carbon atoms and / or all or any of its heteroatoms. A fused heteroaryl radical can contain two to four fused rings when the linker ring is a heteroaromatic ring and the other rings

ΙΜΡΙ

MEXICAN INSTITUTE Dt THE PROPERTY

INDUSTRIAL

<img file="MX342945B_D0184.tif" />

Individuals may be alicyclic, heterocyclic, aromatic, heteroaromatic, or any combination thereof. A non-limiting example of a single ring heteroaryl group includes pyridyl; fused ring heteroaryl groups include benzimidazolyl, quinolinyl, acridinyl; and an un-fused bi-heteroaryl group includes bipyridinyl. Additional examples of heteroaryls include, but are not limited to, furanyl, thienyl, oxazolyl, acridinyl, phenazinyl, benzimidazolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzothioopinyl, isozoyloxy, isoxyloxy, isoxyloxy, isoxyloxy, isoxy, , indazolyl, pyridyl, pyridazyl, pyrimidyl, pyrazinyl, pyrrolyl, pyrazinyl, pyrazolyl, purinyl, phthalazinyl, pteridinyl, quinolinyl, quinazolinyl, quinoxalinyl, triazolyl, tetrazolyl, thiazolyl, triazinyl, thiadiazolyl, and the like, and their oxides, such as, for example, pyridyl-oxide. Illustrative examples of heteroaryl groups include the following residues:

H

H

H

H

<img file="MX342945B_D0185.tif" />

N

<img file="MX342945B_D0186.tif" />

and the like.

ΙΜΡΙ

In one embodiment, heteroaryl

MEXICAN INSTmTO M THE PROPERTY

INDUSTRIAL

<img file="MX342945B_D0187.tif" />

is replaced. Unless otherwise indicated, heteroaryl is unsubstituted.

The term heterocyclyl, as used herein, alone or in combination, refers together to heteroalicyclyl and heteroaryl groups. Herein, whenever the number of carbon atoms in a heterocycle (eg, C1-C6 heterocycle) is indicated, at least one non-carbon atom (the heteroatom) must be present in the ring. Denominations such as C1-C6 heterocycle only refer to the number of carbon atoms in the ring and do not refer to the total number of atoms in the ring. Denominations, such as 4- to 6-membered heterocycle, refer to the total number of atoms contained in the ring (i.e., a four, five, or six-membered ring, where at least one atom is a carbon atom, at least one atom is a hetero atom and the remaining two to four atoms are carbon atoms or hetero atoms). In the case of heterocycles having two or more heteroatoms, said two or more heteroatoms can be the same or different from each other. Non-aromatic heterocyclic groups include groups that have only three ring atoms, while aromatic heterocyclic groups must have at least five atoms

ΙΜΡΙ_

Mexican Institute of Industrial Property

<img file="MX342945B_D0188.tif" />

in the ring. The attachment (ie, the link to a main molecule or the additional substitution) to a heterocycle can occur through a heteroatom or a carbon atom. In one embodiment, the heterocyclyl is substituted. Unless otherwise indicated, the heterocyclyl is unsubstituted.

The terms halogen, halo, or halide, as used herein, alone or in combination, refer to fluoro, chloro, bromo and / or iodo.

Specific pharmaceutical terminology

The term "subject, patient, or individual," as used herein to refer to individuals suffering from a disorder, and the like, encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class of mammals: humans, non-human primates, such as chimpanzees and the other ape and monkey species; farm animals such as cattle, horses, sheep, goats, pigs; pets, such as rabbits, dogs, and cats; laboratory animals, among others, rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.

THE quantity terms

ΙΜ effective

INSTITDTC OF THE

<img file="MX342945B_D0189.tif" />

therapeutically effective or pharmaceutically effective amount, as used herein, refers to an amount of at least one agent or compound that is administered, which is sufficient to treat or prevent the particular disease or condition. The result may be the reduction and / or relief of the signs, symptoms or causes of a disease, or any other desired change in a biological system. For example, an effective amount, for therapeutic purposes, is the amount of the composition comprising a compound, such as that described herein, necessary to provide a clinically significant decrease in disease. An appropriate effective amount in any particular case may be determined using certain techniques, such as, a staged dose increase study.

As used herein, treatment or treating, or mitigating or improving are used interchangeably herein. These terms refer to a way of obtaining beneficial or desired results, among others, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to the eradication or improvement of the underlying disorder being treated.

Likewise, a therapeutic benefit is achieved with

ΙΜΡΙ

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MEXICAN INSTITUTE ¥ * 5 OF PROPERTY £ * «_, INDUSTRIAL X?

eradication or improvement of one or more of the physiological symptoms associated with the underlying disorder so that improvement is seen in the patient, regardless of whether the patient continues to have the underlying disorder. To obtain prophylactic benefit, the compositions can be administered to a patient at risk of developing a particular disease or to a patient who exhibits one or more of the physiological symptoms of a disease, even if the disease has not been diagnosed.

A therapeutic effect, as used herein, comprises a therapeutic benefit and / or a prophylactic benefit as described above. A prophylactic effect includes delaying or withdrawing the onset of a disease or condition, delaying or removing the onset of symptoms of a disease or condition, delaying, stopping or reversing the course of a disease or condition, or any combination thereof.

The term pharmaceutically acceptable, as used herein, refers to a material, such as a carrier or diluent, that does not negate the activity or biological properties of the compounds described herein, and is relatively non-toxic, is that is, the material can be administered to an individual without causing unwanted biological effects or interacting in a harmful way

<img file="MX342945B_D0191.tif" />

IMPI

3 MEXICAN INSTITUTE

OF THE PROPERTY · '

INDUSTRIAL with the components of the composition that contains it.

The term pharmaceutical composition, as used herein, refers to a biologically active compound, optionally mixed with at least one pharmaceutically acceptable chemical component, such as, non-exhaustively, carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents and / or excipients.

The term "carrier" as used herein refers to relatively non-toxic compounds or chemical agents that facilitate the incorporation of a compound into cells or tissues.

The term prodrug is intended to indicate a compound that can be converted under physiological conditions or by solvolysis to a biologically active compound described herein. Accordingly, the term prodrug refers to a precursor of a biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject, but is converted into a compound in vivo, for example, by hydrolysis. The prodrug compound often offers solubility, tissue compatibility, or delayed release benefits in a mammalian organism (see,

Bundgard, Η., Design of Prodrugs (1985), pp. 7-9, 21-24

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0192.tif" />

(Elsevier, Amsterdam). A report on prodrugs is provided in Higuchi, T., et al., Pro-drugs as Novel Delivery Systems, ACS Symposium Series, tome 14 and in

Bioreversible Carriers in Drug Design, ed. Edward B. Roche,

American Pharmaceutical Association and Pergamon Press, 1987, which are incorporated herein in their entirety by this reference. The term prodrug is also intended to include any covalently attached carrier, which releases the active compound in vivo when said prodrug is administered to a mammalian subject. Prodrugs of an active compound, such as those described herein, can be prepared by modifying the functional groups present in the active compound such that the modifications are cleaved, either by routine manipulation or in vivo, to the main active compound. . Prodrugs include compounds where a hydroxy, amino, or mercapto group binds to any group that, when the prodrug of an active compound is administered to a mammalian subject, cleaves to form a free hydroxy, free amino, or free mercapto group, respectively . Examples of prodrugs include, but are not limited to, acyloxy, thioacyloxy, 2-carboalkoxyethyl, disulfide, thiaminal and enol ester derivatives of a nucleic acid modified by a phosphorus atom.

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY pronucleotide or

<img file="MX342945B_D0193.tif" />

reference to a becoming a

The term pro-oligonucleotide or nucleic acid prodrug means oligonucleotide that was modified to prodrug of the oligonucleotide.

Specific terminology on nucleic acids

Natural nucleic acids have a basic phosphate structure; artificial nucleic acids can contain other types of basic structures, but they contain the same bases.

The term nucleotide, as used herein, refers to a monomeric unit of a polynucleotide consisting of a heterocyclic base, a sugar, and one or more phosphate groups. Naturally occurring bases (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are derivatives of purine or pyrimidine, although analogs of Bases of natural or non-natural origin are also included. Naturally occurring sugar is pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), although it is understood that sugar analogues of natural or non-natural origin are also included. Nucleic acids are linked through phosphate linkages to form nucleic acids or polynucleotides, although many other linkages are known in the art (such

IMPI

Mexican Institute of Industrial Property

<img file="MX342945B_D0194.tif" />

as, non-exhaustively, phosphorothioates, boranophosphates and the like). Artificial nucleic acids include APNs (peptide nucleic acids), phosphothionates, and other variants of the basic phosphate structure of natural nucleic acids.

The term nucleoside refers to a moiety where a modified nucleobase or nucleobase is covalently linked to a modified sugar or sugar.

The term sugar refers to a monosaccharide in closed and / or open form. Sugars include, but are not limited to, the residues of ribose, deoxyribose, pentofuranose, pentopyranose and hexopyranose.

The term modified sugar refers to a residue that can replace a sugar. Modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar.

The terms nucleic acid and polynucleotide, as used herein, refer to a polymeric form of nucleotides of any length, both ribonucleotides (RNA) and deoxyribonucleotides (DNA).

These terms refer to the primary structure of molecules, and therefore include double-stranded and single-stranded DNA and double-stranded and single-stranded RNA. These terms include, as equivalents, the

<img file="MX342945B_D0195.tif" />

IMPI

7 MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL RNA and DNA analogs created from nucleotide and modified polynucleotide analogs, such as, but not limited to, methylated and / or coated polynucleotides. The terms include polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from Nglycosides or C-glycosides of modified nucleobases and / or nucleobases; nucleic acids derived from sugars and / or modified sugars and nucleic acids derived from phosphate bridges and / or bridges of modified phosphorous atoms. The term encompasses nucleic acids that contain any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorous atom bridges. Examples include, but are not limited to, nucleic acids containing ribose residues, nucleic acids containing deoxyribose residues, nucleic acids containing ribose and deoxyribose residues, nucleic acids containing ribose residues. and modified ribose. The prefix poly refers to a nucleic acid containing approximately 1 to approximately

10,000 monomeric nucleotide units and where the prefix oligo refers to a nucleic acid containing approximately 1 to approximately 200 monomeric units

IMPI

MEXICAN INSTITUTE OE LA MONEDAD

INDUSTRIAL

<img file="MX342945B_D0196.tif" />

nucleotide. _

The term nucleobase refers to the parts of nucleic acids that are involved in the hydrogen bond that binds one nucleic acid chain to another complementary chain according to the specific sequence. The most common naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T).

The term modified nucleobase refers to a moiety that can replace a nucleobase. The modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the hydrogen bonding property that binds one nucleic acid chain to another according to the specific sequence. A modified nucleobase can pair with any of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting fusion behavior, recognition by intracellular enzymes, or duplex oligonucleotide activity.

The term "chiral reagent" refers to a compound that is chiral or enantiopure and can be used for asymmetric induction in nucleic acid synthesis.

The term chiral ligand or chiral auxiliary makes

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Reference to a moiety that is chiral or enantiopure and controls the stereochemical result of a reaction.

In a condensation reaction, the term condensation reagent refers to a reagent that activates a less reactive site and makes it more susceptible to attack by a nucleophile.

The term blocking group refers to a group that temporarily hides the reactivity of a functional group. Subsequently, the functional group can be hidden by removing the blocking group.

The terms erasing agents, electrophiles of

<td>sulfur,</td><td colspan="2">electrophiles from</td><td colspan="3">selenium refer to</td>

<td>compounds</td><td>than</td><td>they are useful</td><td>at the stage</td><td>of</td><td>modification</td>

<td>used</td><td>for</td><td>Introduce</td><td>the groups</td><td>BH3,</td><td>S and Se,</td>

<td colspan="2">respectively,</td><td colspan="2">for the modification in the</td><td>atom</td><td>phosphorous.</td>

<td colspan="2">The term</td><td colspan="2">rest refers</td><td>yet</td><td>segment o</td>

specific functional group of a molecule. Chemical moieties are often recognized chemical entities, incorporated or added to a molecule.

The term solid support refers to any backing that allows for the mass synthetic production of nucleic acids and that can be reused when needed. As used herein, the term refers to a polymer that is not soluble in the medium.

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INDUSTRIAL JS used in the reaction steps carried out to synthesize nucleic acids and is derived to form reactive groups.

The term "binding moiety" refers to any moiety that is optionally located between the terminal nucleoside and the solid support or between the terminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

A DNA molecule refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in their single-stranded or double-stranded helix form. This term refers to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Therefore, this term includes double-stranded DNA that is found, among others, in linear DNA molecules (eg. , restriction fragments), viruses, plasmids, and chromosomes. By analyzing the structure of particular double-stranded DNA molecules, the sequences can be described herein in accordance with the normal convention of only granting the sequence in the 5 'to 3' direction along the untranscribed strand of DNA (that is, the chain that has a sequence homologated to mRNA).

A sequence coding region of

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DNA coding is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in vivo when placed under the control of the appropriate expression control sequences. The limits of the coding sequence (the open reading frame or ORF) are determined by a start codon at terminal 5¹ (amino) and a translation stop codon at the terminal

3 '(carboxyl). A coding sequence may include, but is not limited to, prokaryotic sequences, eukaryotic mRNA cDNA, eukaryotic DNA genomic DNA sequences (eg, mammalian), and synthetic DNA sequences. A polyadenylation signal and a transcription termination sequence, in general, will be located 3 'with respect to the coding sequence. The term "non-coding sequence" or "non-coding region" refers to regions of a polynucleotide sequence that are not translated into amino acids (eg, 5 'and 3' untranslated regions).

The term reading frame refers to one of six possible reading frames, three in each direction, for the double-stranded DNA molecule. The reading frame that is used determines which codons are used to encode the amino acids within the coding sequence of a DNA molecule.

As used herein, an acid molecule

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antisense nucleic acid comprises a nucleotide sequence that is complementary to a sense nucleic acid encoding a protein, eg, complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence or complementary to the strand of coding a gene. Accordingly, an antisense nucleic acid molecule can be linked by hydrogen to a sense nucleic acid molecule.

<td> 10</td><td>The</td><td>base pair term</td><td>o (pb): a</td><td>association</td><td>of</td>

<td colspan="2">adenine</td><td>(A) with thymine (T) or</td><td>cytosine (C)</td><td>with guanine</td><td>(G)</td>

<td>in</td><td>a</td><td>DNA molecule of</td><td>double chain.</td><td>In RNA,</td><td>I know</td>

substitute thymine for uraciio (U)

As used herein, a codon refers to the three nucleotides that, when transcribed and translated, encode a single amino acid residue, or in the case of UUA, UGA or UAG encode a termination signal.

Codons encoding amino acids are well known in the art and are provided herein for convenience in Table 1.

Table 1

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<img file="MX342945B_D0200.tif" />

Codon utilization table

<td>Codon</td><td>Amino acid</td><td>AA</td><td>Abbrev.</td><td>Codon</td><td>Amino acid or</td><td>AA</td><td>Abbrev.</td>

<td>UUU</td><td>Phenylalani na</td><td>Faith</td><td>F</td><td>UCU</td><td>Serine</td><td>To be</td><td>S</td>

<td>UUC</td><td>Phenylalani na</td><td>Faith</td><td>F</td><td>UCC</td><td>Serine</td><td>To be</td><td>S</td>

<td>UUA</td><td>Leucine</td><td>Leu</td><td>L</td><td>UCA</td><td>Serine</td><td>To be</td><td>S</td>

<td>UUG</td><td>Leucine</td><td>Leu</td><td>L</td><td>UCG</td><td>Serine</td><td>To be</td><td>S</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>CUU</td><td>Leucine</td><td>Leu</td><td>L</td><td>CCU</td><td>Proline</td><td>Pro</td><td>P</td>

<td>CUC</td><td>Leucine</td><td>Leu</td><td>L</td><td>CCC</td><td>Proline</td><td>Pro</td><td>P</td>

<td>CUA</td><td>Leucine</td><td>Leu</td><td>L</td><td>CCA</td><td>Proline</td><td>Pro</td><td>P</td>

<td>CUG</td><td>Leucine</td><td>Leu</td><td>L</td><td>CCG</td><td>Proline</td><td>Pro</td><td>P</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>AUU</td><td>Isoleucine</td><td>lie</td><td>I</td><td>ACU</td><td>Threonine</td><td>Thr</td><td>T</td>

<td>AUC</td><td>Isoleucine</td><td>lie</td><td>I</td><td>ACC</td><td>Threonine</td><td>Thr</td><td>T</td>

<td>AUA</td><td>Isoleucine</td><td>lie</td><td>I</td><td>HERE</td><td>Threonine</td><td>Thr</td><td>T</td>

<td>AUG</td><td>Methionine</td><td>Met</td><td>M</td><td>ACH</td><td>Threonine</td><td>Thr</td><td>T</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>GUU</td><td>Valine</td><td>Val</td><td>V</td><td>GCU</td><td>To the girl</td><td>To</td><td>TO</td>

<td>GUC</td><td>Valine</td><td>Val</td><td>V</td><td>GCC</td><td>To the girl</td><td>To</td><td>TO</td>

<td>GUA</td><td>Valine</td><td>Val</td><td>V</td><td>GCA</td><td>To the girl</td><td>To</td><td>TO</td>

<td>GUG</td><td>Valine</td><td>Val</td><td>V</td><td>GCG</td><td>To the girl</td><td>To</td><td>TO</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>UAU</td><td>Tyrosine</td><td>Tyr</td><td>AND</td><td>UGU</td><td>Cysteine</td><td>Cys</td><td>C</td>

<td>UAC</td><td>Tyrosine</td><td>Tyr</td><td>AND</td><td>UGC</td><td>Cysteine</td><td>Cys</td><td>C</td>

<td>UUA</td><td></td><td>Det Jan ion</td><td></td><td>UGA</td><td></td><td>Dete nation n</td><td></td>

<img file="MX342945B_D0201.tif" />

<td rowspan="2">UAG</td><td rowspan="2"></td><td rowspan="2">Det Jan ion</td><td rowspan="2"></td><td rowspan="2">UGG</td><td rowspan="2">TryptopharT ' or</td><td rowspan="2"></td><td></td>

<td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>CAU</td><td>Histidine</td><td>His</td><td>H</td><td>CGU</td><td>Arginine</td><td>Arg</td><td>R</td>

<td>CAC</td><td>Histidine</td><td>His</td><td>H</td><td>CGC</td><td>Arginine</td><td>Arg</td><td>R</td>

<td>CAA</td><td>Glutamine</td><td>Gln</td><td>Q</td><td>CGA</td><td>Arginine</td><td>Arg</td><td>R</td>

<td>CAG</td><td>Glutamine</td><td>Gln</td><td>Q</td><td>CGG</td><td>Arginine</td><td>Arg</td><td>R</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>AAU</td><td>Asparagine</td><td>Asn</td><td>N</td><td>AGU</td><td>Serine</td><td>To be</td><td>S</td>

<td>AAC</td><td>Asparagine</td><td>Asn</td><td>N</td><td>AGC</td><td>Serine</td><td>To be</td><td>S</td>

<td>AAA</td><td>Lysine</td><td>Lys</td><td>K</td><td>AGA</td><td>Arginine</td><td>Arg</td><td>R</td>

<td>AAG</td><td>Lysine</td><td>Lys</td><td>K</td><td>AGG</td><td>Arginine</td><td>Arg</td><td>R</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>GAU</td><td>Aspartate</td><td>Asp</td><td>D</td><td>GGU</td><td>Glycine</td><td>Gly</td><td>G</td>

<td>GAC</td><td>Aspartate</td><td>Asp</td><td>D</td><td>GGC</td><td>Glycine</td><td>Gly</td><td>G</td>

<td>GAA</td><td>Glutamate</td><td>Glu</td><td>AND</td><td>GGA</td><td>Glycine</td><td>Gly</td><td>G</td>

<td>GAG</td><td>Glutamate</td><td>Glu</td><td>AND</td><td>GGG</td><td>Glycine</td><td>Gly</td><td>G</td>

As used herein, a wobbly position refers to the third position of a codon. Mutations in a DNA molecule within the wobbly position of a codon, in some embodiments, result in imperceptible or conservative mutations at the amino acid level. For example, there are four codons that encode Glycine, that is, GGU, GGC, GGA and GGG, therefore, the mutation of any nucleotide in the wobbly position, to any other nucleotide, does not give

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A change at the amino acid level of the encoded protein results and is therefore an imperceptible substitution.

Accordingly, an undetectable substitution or undetectable mutation is one in which a nucleotide within a codon is modified but this does not result in a change in the amino acid residue encoded by the codon. Examples include mutations at the third position of a codon, as well as at the first position of certain codons, such as in the CGG codon which, when mutated to AGG, still encodes Arg.

The terms gene, recombinant gene, and gene construct, as used herein, refer to a DNA molecule or to a residue of a DNA molecule that encodes a protein or a portion thereof. The molecule of

DNA can contain an open reading frame that encodes the protein (such as exon sequences) and can also include intron sequences. The term intron, as used herein, refers to a DNA sequence present in a given gene that is not translated into a protein and is found in some, but not all, cases of exons. It may be desirable for the gene to be operably linked to (or comprise) one or more promoters, enhancers, repressors and / or other regulatory sequences, to modulate the activity or expression of the gene, as is well known in the art.

industrial

As used herein, a compleuienlaf'lu · DNA or cDNA includes recombinant polynucleotides synthesized by reverse transcription of the mRNA and from which the intermediate sequences (introns) have been removed.

Homology, identity or similarity refer to the sequence similarity between two nucleic acid molecules. Homology and identity can be determined by comparing a position in each sequence, which can be aligned for the purposes of their comparison. When an equivalent position in the compared sequences is occupied by the same base, the molecules are identical at that position; When the equivalent site occupied by the same nucleic acid residue, or a similar one (eg, similar in terms of its spherical and / or electronic nature), the molecules can be called homologous (similar) in said position. The expression as a percentage of homology / similarity or identity refers to a function of the number of identical or similar nucleic acids at the positions shared by the compared sequences. An unrelated or non-homologous sequence shares less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with a sequence described herein. To the

<img file="MX342945B_D0203.tif" />

9Ί i ívi ri A * compare two sequences, the absence of residues (^!S ¥ ifÍ ¥ íóá¿ or nucleic acids) or the presence of resTüúóy cUllulunales also decreases identity and homology / similarity.

The term homology describes a mathematical comparison of sequence similarity that is used to identify genes with similar motifs or functions. The nucleic acid sequences described herein can be used as a query sequence to search public databases, for example, to identify other family members, related sequences, or homologs. Such searches can be carried out using the NBLAST and XBLAST (version 2.0) programs from Altschul, et al. (1990) J. Mol. Biol.

215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. To obtain incomplete alignments for comparison purposes, the Gapped BLAST can be used, as described in Altschul et al., (1997) Nucleic Acids

Res. 25 (17): 3389-3402. When using the BLAST and Gapped programs

BLAST, the default parameters of the corresponding programs can be used (eg XBLAST and BLAST) (See www.nebí.nlm.nih.gov).

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As used herein, identity makes

<img file="MX342945B_D0204.tif" />

reference to the percentage of identical nucleotide residues at the corresponding positions in two or more sequences, when the sequences are aligned to maximize the coincidence of the sequences, that is, taking into account gaps and insertions. Identity can be easily calculated by known methods, among others, those described in (Computational Molecular

Biology, Lesk, AM, ed., Oxford University Press, New

York, 1988; Biocomputing: Informatics and Genome Projects,

Smith, DW, ed., Academic Press, New York, 1993; Computer

Analysis of Sequence Data, Part I, Griffin, AM, and Griffin,

HG, eds., Humana Press, New Jersey, 1994; Sequence

Analysis in Molecular Biology, von Heinje, G., Academic

Press, 1987; and Sequence Analysis Primer, Gribskov, M. and

Devereux, J., eds., M Stockton Press, New York, 1991; and

Carillo, H., and Lipman, D., SIAM J. Applied Math. , 48: 1073 (1988). The methods for determining identity are designed to provide the best match between the analyzed sequences. In addition, the methods for determining identity are encoded in publicly available computer programs. Methods of computer programs to determine the identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids

INSTITUTO MI DE LA PR (. _ IND

<img file="MX342945B_D0205.tif" />

12 (1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S¹. F.

et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al.

Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available at NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda,

Md. 20894; Altschul, S., et al. , J. Mol. Biol. 215: 403-410 (1990). Smith Waterman's well-known algorithm can also be used to determine identity.

A heterologous region of a DNA sequence is an identifiable segment of DNA within a larger DNA sequence that is not associated with the largest sequence in nature. Accordingly, when the heterologous region encodes a mammalian gene, in general, the gene may be flanked by DNA that does not flank mammalian genomic DNA in the genome of the original organism. Another example of a heterologous coding sequence is a sequence in which the coding sequence itself is not found in nature (for example, a cDNA where the genomic coding sequence contains introns or synthetic sequences that possess different codons or motifs. to the unmodified gene). Allelic variations or naturally occurring mutational events do not generate a heterologous region of DNA as defined herein.

<img file="MX342945B_D0206.tif" />

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The term transitional mutations refers to the basic changes in a DNA sequence where a pyrimidine (cytidine (C) or thymidine (T) is replaced by another pyrimidine, or a purine (adenosine (A) or guanosine (G) is replaced by another purine.

<td>The</td><td>term mutations</td><td colspan="2">transversal</td><td colspan="2">make</td>

<td>reference</td><td>to basic changes</td><td>in</td><td>a sequence</td><td>of</td><td>DNA</td>

<td>where a</td><td>pyrimidine (cytidine</td><td>(C)</td><td>or thymidine</td><td>(T)</td><td>is</td>

<td>replaced</td><td colspan="2">for a purine (adenosine</td><td>(A) or guanosine</td><td>(G)</td><td>, or</td>

a purine is replaced by a pyrimidine.

Nucleic acid prodrugs comprising a chiral residue of X-phosphonate

The general principles of prodrug design are detailed by Bundgard (Design and Application of Prodrugs.

In a Textbook of Drug Design and Development; KrogsgaardLarsen, P., Bundgard, H., Eds .; Harwood: Reading, UK, 1991).

One strategy to improve the pharmaceutical properties of molecules with beneficial biological activity but little pharmaceutical properties is to administer the molecule of interest as a derivative of the prodrug. These prodrugs may exhibit one or more of the following properties: increased oral bioavailability, increased cell permeability, increased water solubility, reduced first-pass effect,

101

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PROPERTY, INDUSTRIAL increased stability, active transport of intestinal transporters or the possibility of avoiding efflux transporters, when compared to the main molecule.

Oligonucleotides possess various pharmaceutical properties that can be improved by applying prodrug strategies. In particular, oligonucleotides are rapidly degraded by nucleases and exhibit poor cellular absorption across the cytoplasmic cell membrane (Poijarvi-Virta et al.,

Curr. Med. Chem. (2006), 13 (28); 3441-65; Wagner et al. , Med.

Res. Rev. (2000), 20 (6): 417-51, - Peyrottes et al. , Mini Rev.

Med. Chem. (2004), 4 (4): 395-408, - Gosselin et al. , (nineteen ninety six),

43 (1): 196-208, - Bologna et al. , (2002), Antisense &amp; Nucleic

Acid Drug Development 12: 33-41). In one example, Vives et al., (Nucleic Acids Research (1999), 27 (20): 4071-76) found that tere-butyl SATE pro-oligonucleotides exhibit a considerable increase in cell penetration compared to the oligonucleotide principal.

In some embodiments, the prodrug moiety is selectively removed by esterases, nucleases, or a cytochrome P450 enzyme, which include, but are not limited to, those listed below.

<td>Family</td><td>Gen</td><td colspan="2"> 102</td><td>Family</td><td>MEXICAN INSTITUTE ÍJ • E PROPERTY • tNtnisTWAf ··· '· Gen</td>

<td>CYP1</td><td>CYP1A1,</td><td>CYP1A2,</td><td>CYP1B1</td><td>CYP17</td><td>CYP17A1 ---- -</td>

<td></td><td>CYP2A6,</td><td>CYP2A7,</td><td></td><td>CYP19</td><td>CYP19A1</td>

<td></td><td>CYP2A13</td><td>, CYP2B6,</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>CYP20</td><td>CYP20A1</td>

<td></td><td>CYP2C8,</td><td>CYP2C9,</td><td></td><td></td><td></td>

<td>CYP2</td><td>CYP2C18</td><td>, CYP2C19</td><td>z</td><td>CYP21</td><td>CYP21A2</td>

<td></td><td>SCYP2D6,</td><td>CYP2E1,</td><td>CYP2F1, '</td><td>CYP24</td><td>CYP24A1</td>

<td></td><td>CYP2J2,</td><td>CYP2R1,</td><td>CYP2S1,</td><td></td><td></td>

<td></td><td>CYP2U1,</td><td>CYP2W1</td><td></td><td>CYP2 6</td><td>CYP26A1, CYP26B1,</td>

<td></td><td></td><td></td><td></td><td></td><td>CYP26C1</td>

<td></td><td>CYP3A4,</td><td>CYP3A5,</td><td>CYP3A7,</td><td></td><td></td>

<td>CYP3</td><td>CYP3A43</td><td></td><td></td><td></td><td>CYP27A1</td>

<td></td><td></td><td></td><td></td><td></td><td>(biosynthesis of</td>

<td></td><td>CYP4A11</td><td>, CYP4A22</td><td>z</td><td></td><td>bile acid),</td>

<td></td><td>CYP4B1,</td><td>CYP4F2,</td><td>CYP4F3,</td><td></td><td>CYP27B1</td>

<td>CYP4</td><td>CYP4F8,</td><td>CYP4F11,</td><td></td><td>CYP2 7</td><td>(1-alpha hydroxylase</td>

<td></td><td>CYP4F12</td><td>, CYP4F22</td><td>z</td><td></td><td>^ vitamin D3, active</td>

<td></td><td>CYP4V2,</td><td>CYP4X1,</td><td>CYP4Z1</td><td></td><td>vitamin D3),</td>

<td>CYP5</td><td>CYP5A1</td><td></td><td></td><td></td><td>CYP27C1 (function</td>

<td></td><td></td><td></td><td></td><td></td><td>unknown)</td>

<td>CYP7</td><td>CYP7A1,</td><td>CYP7B1</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>CYP3 9</td><td>CYP39A1</td>

<td></td><td>CYP8A1</td><td>(synthase</td><td>of</td><td></td><td></td>

<td>ΓΎΡΑ</td><td colspan="2">prostacyclin),</td><td>CYP8B1</td><td>CYP4 6</td><td>CYP46A1</td>

<td></td><td colspan="2">(biosynthesis of</td><td>acid</td><td></td><td>CYP51A1 (lanosterol</td>

<td></td><td>bile)</td><td></td><td></td><td>CYP51</td><td>: 14-alpha demethylase)</td>

<td></td><td>! CYPllAl</td><td>, CYP11B1</td><td></td><td></td><td></td>

<td>CYPll</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>; CYP11B2</td><td></td><td></td><td></td><td></td>

In some embodiments, the prodrug is withdrawn when the prooligonucleotide has not yet been transported through the cell membrane. In other modalities, the prodrug is removed from the prooligonucleotide only after being transported through the cell membrane. Alternatively, the prodrug is withdrawn only after being transported to an organelle within the cell. In some embodiments, the remaining prodrug is removed through nonenzymatic deletion, which includes

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exhaustive, the spontaneous reduction within the cell.

Nucleic acid prodrugs are described herein that comprise modification of a chiral X-phosphonate, where the modification improves one or more physicochemical, pharmacokinetic, or pharmacodynamic properties of the nucleic acid. A prodrug moiety is connected to an oxygen or sulfur atom that is connected to the phosphorus atom of a nucleotide phosphonate or phosphothiorate group. The prodrug moiety includes, but is not limited to, S-acyl10 2-thioethyl, acyloxy, thioacyloxy, 2-carboalkoxyethyl, disulfide, thiaminal, and enol ester derivatives.

In one embodiment, the prodrug residue is a residue of

S-acyl-2-thioethyl having the following structure:

<img file="MX342945B_D0209.tif" />

where Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl. In some embodiments, Rll is methyl, ethyl, or cyclopropyl.

In other embodiments, the prodrug residue is an acyloxy residue having the following structure:

where Rll is alkyl, aryl, heteroaryl, heterocyclyl

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<img file="MX342945B_D0210.tif" />

or cycloalkyl and R12 is hydrogen or alkyl. In some embodiments, Rll is methyl and R12 is hydrogen.

Alternatively, the prodrug residue is a thioacyloxy residue having the following structure:

<img file="MX342945B_D0211.tif" />

where Rll is alkyl, aryl, heteroaryl, heterocyclyl or cycloalkyl and R12 is hydrogen or alkyl. In some embodiments, Rll is methyl and R12 is hydrogen.

The invention also provides a prodrug moiety.

2-carboalkoxyethyl having one of the following structures:

<img file="MX342945B_D0212.tif" />

_or Or where RIO is an alkyl group having 1 to 4 carbon atoms. In some embodiments, RIO is methyl or ethyl.

In still other modalities, the prodrug residue is a disulfide residue that has the following structure:

Ace-⁸where Rll is alkyl, aryl, heteroaryl, heterocyclyl or cycloalkyl. In some embodiments, Rll is methyl, ethyl, or benzyl.

<img file="MX342945B_D0213.tif" />

MEXICAN INSTITUTE OF LA MONEAD

105

INDUSTRIAL

In additional modalities, the prodrug residue is a thioacetal residue that has the following structure:

<img file="MX342945B_D0214.tif" />

where RIO is an alkyl group having 1 to 4 carbon atoms; and Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl. In some embodiments RIO is methyl, and Rll is methyl or phenyl.

The invention also provides enol ester prodrugs having one of the following structures:

<img file="MX342945B_D0215.tif" />

and^s^^z^ o&gt; | -R₁₁ or

enol ester C4 enol ester C3 where Rll is alkyl, aryl, heteroaryl, heterocyclyl or cycloalkyl. In some embodiments, the C3 ester enol prodrug moiety or the C4 ester enol prodrug moiety is in the cis form. In some embodiments of the C3 ester enol prodrug moiety or the C4 ester enol prodrug moiety, Rll is methyl, ethyl or phenyl.

In one embodiment, the prodrug moiety is a trialkylammonioethyl moiety having one of the following

106

<img file="MX342945B_D0216.tif" />

structures:

IMPI

Mexican Institute of Industrial Property

<img file="MX342945B_D0217.tif" />

© .Me γθ

Me © -Me i? Me

I

In one embodiment, the prodrug moiety is an alkylhydroxamate moiety having one of the following structures:

<img file="MX342945B_D0218.tif" />

In one embodiment, the prodrug residue is an acylhydroxamate residue having one of the following structures:

<img file="MX342945B_D0219.tif" />

One embodiment provides a nucleic acid prodrug having the following structure:

<img file="MX342945B_D0220.tif" />

Formula 1

107

<img file="MX342945B_D0221.tif" />

where Rl is -OH, -SH, -NRdRd, -N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, aryl-Yl-, heteroaryl-Yl-, -P (O) (Re) 2,

-HP (O) (Re), -ORa or -SRc;

Yl is O, NRd, S or Se;

Ra is a blocking group;

Re is a blocking group;

each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (0) (Re) 2 or -HP (O) (Re);

each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2-, alkenylY2-, alkynyl-Y2-, aryl-Y2- or heteroaryl-Y2-, or a cation that is Na + 1, Li + 1 or K + l;

Y2 is 0, NRd or S;

each instance of R2 is independently hydrogen,

-OH, -SH, -NRdRd, -N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, arylYl-, heteroaryl-Yl-, -ORb or -SRc, where Rb is a blocking group;

each instance of Ba is independently blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;

at least one instance of X is - OCH2CH2S-S (0) 2R10, 108 *ⁿ^^ to_{M £ XICa}* .

Ot LA EJtOp | £ Q_TO.,

INOUSTK ^ yes

OCH2CH2S-SCH2CH2QH,

-OCH2CH2CO2H,

<img file="MX342945B_D0222.tif" />

ΑβΛΛ

<img file="MX342945B_D0223.tif" />

<img file="MX342945B_D0224.tif" />

<img file="MX342945B_D0225.tif" />

<img file="MX342945B_D0226.tif" />

<img file="MX342945B_D0227.tif" />

<img file="MX342945B_D0228.tif" />

<img file="MX342945B_D0229.tif" />

<img file="MX342945B_D0230.tif" />

<img file="MX342945B_D0231.tif" />

&lt;j&gt; me

<img file="MX342945B_D0232.tif" />

OR

O o

<img file="MX342945B_D0233.tif" />

OR

<img file="MX342945B_D0234.tif" />

0

MEXICAN INSTITUTE

OE THE PROPERTY

INDUSTRIA L

109

<img file="MX342945B_D0235.tif" />

R3 is hydrogen, a blocking group? un — yeotüi da. binding connected to a solid support or a binding moiety connected to a nucleic acid;

RIO is an alkyl group having 1 to 4 carbon atoms;

Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl;

R12 is hydrogen or alkyl;

<td></td><td>z</td><td>is</td><td>S</td><td>u 0;</td><td></td>

<td> 10</td><td>what</td><td>is</td><td> 0,</td><td>1 or 3;</td><td></td>

<td></td><td>w</td><td>is</td><td> 1,</td><td>2. 3. 4 t -s 1 ~ f</td><td>5 or 6;</td>

R15 and R16 are independently hydrogen or methyl;

R17 is selected from alkyl, aryl, or a CH2CH = CH2;

R18 is selected from N (CH3) 2

<img file="MX342945B_D0236.tif" />

<img file="MX342945B_D0237.tif" />

<img file="MX342945B_D0238.tif" />

n is an integer from 1 to about 200.

In one aspect, the invention provides a nucleic acid prodrug 20 having the following structure:

<img file="MX342945B_D0239.tif" />

Formula 1

ΙΜΡΙ

<img file="MX342945B_D0240.tif" />

η η Π MEXICAN INSTITUTE fl ** (

INDUSTRIAL PROPER TY U *&gt; 57 ^ where Rl is -OH, -SH, -NRdRd, -N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, aryl -Yl-, heteroaryl-Yl-, -OP (O) (Re) 2,

HP (O) (Re), -ORa or -SRc;

<td> 5</td><td>Yl</td><td>is</td><td> 0,</td><td>NRd,,</td><td>S or Se;</td>

<td></td><td>Ra</td><td>is</td><td>a</td><td>group</td><td>blocking;</td>

<td></td><td>Rc</td><td>is</td><td>a</td><td>group</td><td>blocking;</td>

each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (0) (Re) 2 or -HP (O) (Re);

each instance of Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y2-, alkenylY2-, alkynyl-Y2-, aryl-Y2- or heteroaryl-Y2-, or a cation that is Na + 1, Li + 1 or K + l;

Y2 is 0, NRd or S;

each instance of R2 is independently hydrogen, -OH, -SH, -NRdRd, -N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, arylYl-, heteroaryl-Yl- , -ORb or -SRc, where Rb is a blocking group;

each instance of Ba is independently blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;

lll

<img file="MX342945B_D0241.tif" />

each instance of X

<img file="MX342945B_D0242.tif" />

<img file="MX342945B_D0243.tif" />

<img file="MX342945B_D0244.tif" />

<img file="MX342945B_D0245.tif" />

R3 is hydrogen, a blocking group, a bonding moiety connected to a solid support, or a bonding moiety connected to a nucleic acid;

RIO is an alkyl group having 1 to 4 carbon atoms;

112

IMPI

MBXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0246.tif" />

Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl;

R12 is hydrogen or alkyl; and n is an integer from 1 to about 200.

In one aspect, the invention provides a nucleic acid prodrug having the following structure:

<img file="MX342945B_D0247.tif" />

Formula 2 where Rl is -OH, -SH, -NRdRd, -N3, halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, aryl-Yl-, heteroaryl-Yl -, -OP (O) (Re) 2,

HP (O) (Re), -ORa or -SRc;

Yl is 0, NRd, S or Se;

Ra is a blocking group;

Rc is a blocking group;

each instance of Rd is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (0) (Re) 2 or -HP (O) (Re);

each instance of Re is independently hydrogen,

IΜ ΡI i 1; MEXICAN INSTITUTE

J- -L OF PROPERTY

INDUSTRIAL alkyl, aryl, alkenyl, alkynyl, alkyl-Y2-, alkenylY2-, alkynyl-Y2-, aryl-Y2- or heteroaryl-Y2-, or a cation which is Na + 1, Li + 1 or K + l;

Y2 is 0, NRd or S;

each instance of R2 is independently hydrogen,

-OH, -SH, -NRdRd, -N3, halogen, alkyl, alkenyl, alkynyl, alkyl-Yl-, alkenyl-Yl-, alkynyl-Yl-, arylYl-, heteroaryl-Yl-, -ORb or -SRc, where Rb is a blocking group;

each instance of Ba is independently blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase; rq □ q each instance of X is'^R11

<img file="MX342945B_D0248.tif" />

?¹² or ?¹² N

ΑΛΑ ,, ΥΆΎ ,,

V^s^ VAo γ ° —γ ° 'ρ, ο oo '10

V

<img file="MX342945B_D0249.tif" />

<img file="MX342945B_D0250.tif" />

&lt;¡&gt; Me N

OR

I

I

I

I \ ^I V ^ V ^ Me

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY (jY ^ Me

Ά^ΝχΜβ

R3 is hydrogen, a blocking group, a bonding moiety connected to a solid support, or a bonding moiety connected to a nucleic acid;

RIO is an alkyl group having 1 to 4 carbon atoms;

Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl;

R12 is hydrogen or alkyl; and n is an integer from 1 to about 200.

A further embodiment provides the nucleic acid prodrug of Formula 1 or Formula 2, where each residue of

X of the nucleic acid prodrug is independently selected from -OCH2CH2S-S (0) 2R10, -OCH2CH2SSCH2CH2OH, -OCH2CH2CO2H,

<img file="MX342945B_D0251.tif" />

<img file="MX342945B_D0252.tif" />

<img file="MX342945B_D0253.tif" />

<img file="MX342945B_D0254.tif" />

-¡I

OR

<img file="MX342945B_D0255.tif" />

<img file="MX342945B_D0256.tif" />

X ^K10

<img file="MX342945B_D0257.tif" />

<img file="MX342945B_D0258.tif" />

IMPI

<img file="MX342945B_D0259.tif" />

115

<img file="MX342945B_D0260.tif" />

You

<img file="MX342945B_D0261.tif" />

River

<img file="MX342945B_D0262.tif" />

OR

<img file="MX342945B_D0263.tif" />

<img file="MX342945B_D0264.tif" />

<img file="MX342945B_D0265.tif" />

© Me

I

<img file="MX342945B_D0266.tif" />

&lt;!)

N. _ΛI

<img file="MX342945B_D0267.tif" />

<td>R3</td><td>is</td><td>hydrogen,</td><td>a blocking group, a</td><td>rest</td><td>of</td>

<td colspan="3">link connected to a</td><td>solid support or a rest</td><td colspan="2">link</td>

<td>connected</td><td colspan="3">to a nucleic acid;</td><td></td><td></td>

<td>RIVER</td><td>is</td><td>a group</td><td>alkyl that has from 1 to 4</td><td>atoms</td><td>of</td>

<td>carbon;</td><td></td><td></td><td></td><td></td><td></td>

<td>Rll</td><td>is</td><td>I rent,</td><td colspan="2">aryl, heteroaryl, heterocyclyl</td><td>or</td>

cycloalkyl;

116

R12 is hydrogen or alkyl;

IMPI

MEXICAN INSTITUTE OE INDUSTRIAL PROPERTY

Z is SUO;

q is 0, 1 or 3;

w is 1, 2, 3, 4, 5, or 6;

R15 and R16 are independently hydrogen or methyl;

R17 is selected from alkyl, aryl, or a CH2CH = CH2; and

R18 is selected from N (CH3) 2,

<img file="MX342945B_D0268.tif" />

N.

<td>In some</td><td>modalities,</td><td>n is a number</td><td colspan="2">integer of 1</td>

<td>approximately</td><td>fifty; 1 a</td><td>approximately</td><td> 40;</td><td> 1</td>

<td>approximately</td><td>30; 1 a</td><td>approximately</td><td> 25;</td><td> 1</td>

<td>15 approximately</td><td>twenty; 1 a</td><td>approximately</td><td> 15;</td><td>O 1</td>

about 10.

One embodiment provides a non-racemic pro-oligonucleotide where the pro-oligonucleotide is a 2-5A analog, having a structure of the following formula:

<img file="MX342945B_D0269.tif" />

HO OH

Formula A3-1

117

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0270.tif" />

where X is any of the prodrug residues described herein.

In some embodiments, the non-racemic pro-oligonucleotide is a 2-5A analog that has the following structure:

<img file="MX342945B_D0271.tif" />

where Rll is alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl.

In one embodiment, the non-racemic pro-oligonucleotide is a 2-5A analog having the following structure:

<img file="MX342945B_D0272.tif" />

Formula A3-3

118

ΙΜΡΙ

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0273.tif" />

EXAMPLES OF SUMMARY METHODS

General discussion about the synthesis methods of nucleic acid prodrugs that comprise a chiral Xphosphonate moiety

The methods described herein provide efficient synthesis of phosphorus atom modified nucleic acid prodrugs where the stereochemical configuration at a phosphorus atom is controlled, thereby producing a stereodefined oligonucleotide. While the examples of synthesis methods described herein provide a 3'-5 'nucleotide link, the 2'-5' nucleotide link is also contemplated.

The pro-oligonucleotides of the invention can be synthesized by modifying either a chiral phosphorothioate or a chiral Hphosphonate of a nucleotide or nucleic acid.

An S-acyl-2-thioethyl pronucleotide can be synthesized from a nucleic acid or nucleotide comprising one as shown in the following

Chiral H-phosphonate scheme:

Scheme 1

<img file="MX342945B_D0274.tif" />

OR³

<img file="MX342945B_D0275.tif" />

In some modalities, Rl is -0P (0) (Re) 2, dSftl3e^{To the}Re is

119

IMPI

MEXICAN INSTITUTE OF LA PRUFIEDA »

<img file="MX342945B_D0276.tif" />

-OR

O The chiral H-phosphonate is treated with N-chlorosuccinimide and then reacted with Sacyl-2-thioethyl alcohol to produce an S-acyl-2-thioethyl prodrug. The protecting groups present in Rl, R2 and / or R3 can be removed later.

An acyloxy nucleic acid prodrug can be synthesized from a nucleic acid or nucleotide comprising a chiral H-phosphonate as shown in the following scheme: Scheme 2

<img file="MX342945B_D0277.tif" />

<img file="MX342945B_D0278.tif" />

<img file="MX342945B_D0279.tif" />

R²

The chiral H-phosphonate is treated with N-chlorosuccinimide and then reacted with a hydroxymethyl acetate compound to produce an acyloxy prodrug. The protecting groups present in Rl, R2 and / or R3 can be removed later.

A thioacyloxy nucleic acid prodrug can be synthesized from a nucleic acid or nucleotide comprising a chiral phosphorothioate as shown in the following scheme:

120

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0280.tif" />

Scheme 3 R¹-i π Ba

V cT''O w

R²

Ba pJ

R²

OR³

R11 O Cl

Ri 1

Y ° \ -s _ror OC? "Ό

R²

V7

Ba

R²

OR³

Phosphorothioate is treated with an acyloxy chloromethyl compound to produce an acyloxy prodrug. The protecting groups present in Rl, R2 and / or R3 can be removed later.

A 2-carboalkoxyethyl nucleic acid prodrug can be synthesized from a nucleic acid or nucleotide comprising a chiral phosphorothioate as shown in the following scheme:

<img file="MX342945B_D0281.tif" />

MeO '

<img file="MX342945B_D0282.tif" />

The deprotonated chiral phosphorothioate is reacted with an alkyl acrylate to produce a pronucleotide of

2-carboalkoxyethyl. The protecting groups present in Rl,

R2 and / or R3 can be removed later.

A nucleic acid disulfide prodrug can be synthesized from a nucleic acid or nucleotide that

121 A chiral phosphorothioate comprises the following scheme:

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0283.tif" />

as shown in

<img file="MX342945B_D0284.tif" />

<img file="MX342945B_D0285.tif" />

The deprotonated chiral phosphorothioate is reacted with a dialkyl sulfide to produce an alkyl disulfide pronucleotide. The protecting groups present in Rl, R2 and / or R3 can be removed later.

A thioacetal nucleic acid prodrug can be synthesized from a nucleic acid or nucleotide comprising a chiral phosphorothioate as shown in the following scheme:

<img file="MX342945B_D0286.tif" />

R- | qO

Rio n

<img file="MX342945B_D0287.tif" />

A 1,1-dialkyloxy 3-acyloxy propane is reacted with trimethylsilyltriflate and the deprotonated chiral phosphorothioate is then added to the reaction mixture to produce a thioacetal pronucleotide. The groups

122

IMPI

MEXICAN INSTITUTE D £ LA PROF1FOAO industrial

<img file="MX342945B_D0288.tif" />

Protectors present in Rl, R2 and / or R3 can be removed later.

A C3 ester enol nucleic acid prodrug can be synthesized from a nucleic acid or nucleotide comprising a chiral phosphorothioate as shown in the following scheme:

<img file="MX342945B_D0289.tif" />

θ R11

OR

<img file="MX342945B_D0290.tif" />

The deprotonated chiral phosphorothioate is reacted with a compound (E) -3-chloro-1-acyloxy-prop-l-ene to produce the C3 ester enol nucleic acid prodrug. The protecting groups present in Rl, R2 and / or R3 can be removed later.

A C4 ester enol nucleic acid prodrug can be synthesized from a nucleic acid or nucleotide comprising a chiral phosphorothioate as shown in the following scheme:

<img file="MX342945B_D0291.tif" />

Cl

<img file="MX342945B_D0292.tif" />

123

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL MONEDAD

<img file="MX342945B_D0293.tif" />

The deprotoned chiral phosphorothioate is reacted with a compound (E) -3-chloro-1-acyloxy-but-l-ene to produce the C3 enol ester nucleic acid prodrug. The protecting groups present in Rl, R2 and / or R3 can be removed later.

In some embodiments, the nucleic acid comprising a chiral phosphorothioate or a chiral H-phosphonate is synthesized as described herein. In other embodiments, other synthesis methods can be used to provide the nucleic acid comprising a chiral phosphorothioate or chiral Hphosphonate.

Scheme 9. Synthesis of chiral phosphorothioate precursors of the oligonucleotides of the prodrug of the invention. (Route A)

<img file="MX342945B_D0294.tif" />

Formula 1'

<img file="MX342945B_D0295.tif" />

chain lengthening cycle

<img file="MX342945B_D0296.tif" />

I cover you

<img file="MX342945B_D0297.tif" />

124

ΙΜΡΙ

Mexican Institute of Industrial Property

<img file="MX342945B_D0298.tif" />

Reaction of a molecule comprising an achiral H-phosphonate residue of Formula 2 with a nucleoside comprising a nucleophilic residue of Formula IV results in the formation of a condensed intermediate (V);

which is converted to a nucleic acid comprising a chiral X'-phosphonate moiety which can be further modified to produce the oligonucleotide of the prodrug of Formula I comprising a chiral X-phosphonate moiety. Synthesis of the condensed intermediate comprises the steps of (a) activating the compound of Formula 2 with a condensing agent to form intermediate II, (b) reaction with a chiral reagent to form intermediate III, followed by (c) reaction with a compound of Formula IV.

The condensed intermediate can be converted to a nucleic acid comprising a chiral X 'phosphonate moiety of Formula 1' by coating the chiral auxiliary with a moiety A, which is an acyl, aryl, alkyl, aralkyl or silyl moiety and modifying the phosphorus to enter J, which is S, Se or

BH3, thereby producing a compound of Formula VII.

The compound of Formula VII can be converted to the compound of Formula 1 ', where X' is S, Se, or BH3, and n is 1, cleaving the chiral reagent and unblocking blocking groups and cleaving them from the solid support if desired.

Alternatively, the compound of Formula VII is subjected

IMPI ² -&gt; MEXICAN INSTITUTE

FROM 'INDUSTRIAL PROPERTY&gt; ^ ΤΚΓ to chain elongation by unlocking the 5' end and repeating coupling steps to produce a condensed intermediate as previously performed. The steps of coating, modification, unlocking and chain lengthening are repeated until the desired n is obtained.

At that time, chiral reagents are cleaved at each phosphonate, the remaining blocking groups are cleaved, including cleavage from a solid support, if desired, to produce the compound of Formula 1 ', where X' is

S, Se or BH3, and n is greater than or equal to 2 and less than about 200. The compound of Formula 1 ', where X' is

S is then converted by methods described herein to form the pro-oligonucleotide compound of

Formula 1.

Modifying agents used to enter S, Se or

BH2 in chiral phosphorus from the condensed intermediate V in the

Route A.

In some embodiments, the modifying agent is a sulfur electrophile, selenium electrophile, or boronation agent. In some embodiments, the sulfur electrophile is a compound that has one of the following formulas:

S8 (Formula B), Z10-SS-Z11 or Z1Q-SX-Z11, where Z10 and Zll are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic,

<img file="MX342945B_D0299.tif" />

MIXICAN INSTITUTE OF INDUSTRIAL PROPERTY

125 cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide or thiocarbonyl or Z10 and Zll are taken together to form a 3- to 8-membered alicyclic or heterocyclic ring, which may be substituted or unsubstituted; X is S02, O or NRf;

and Rf is hydrogen, alkyl, alkenyl, alkynyl, or aryl. In other embodiments, the sulfur electrophile is a compound of Formula B, C, D, E, or F:

<td>Sg</td><td>nh₇Λ • i</td><td>OEt TO one</td><td></td>

<td>Formula b</td><td>Formula ^c</td><td>Formula E&gt;</td><td>Formula h</td>

<img file="MX342945B_D0300.tif" />

In other embodiments, the sulfur electrophile is

Formula F, Formula E or Formula B.

In some embodiments, the selenium electrophile is a compound that has one of the following formulas:

Se (Formula G), Z10-Se-Se-Zll or Z10-Se-X-Zll, where Z10 and Zll are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide or thiocarbonyl or Z10 and Zll are taken together to form a 3-8 membered alicyclic or heterocyclic ring, which

127

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0301.tif" />

they can be substituted or

NRf; and Rf is hydrogen, aryl.

unsubstituted;

X is S02,

S, 0 O alkyl, alkenyl, alkynyl or

In other embodiments, the selenium electrophile is a

<td colspan="7">Compound of Formula G, Η, I, J, KqL.</td>

<td></td><td></td><td></td><td></td><td rowspan="2">CQ *</td><td>Ph</td><td rowspan="2">N (/ \ e-Se_pN</td>

<td>I know</td><td>KSeCN</td><td>Ph-</td><td>-P — P (| ¿H</td><td>'be-be Ph</td>

<td>Formula g</td><td>Formula h</td><td colspan="2">Formula l</td><td>Formula J</td><td>Formula k</td><td>Formula L</td>

In some embodiments, the selenium electrophile is

Formula G or Formula L.

In some embodiments, the erasing agent is boran-N, N-diisopropylethylamine (BH3-DIPEA), boran-pyridine (BH3-Py), boran-2-chloropyridine (BH3-CPy), boran-aniline (BH3-An) , boran-tetrahydrofuran (BH3-THF) or borandimethylsulfide (BH3-Me2S), añilina-cyanoborane, triphenylphosphine-carboalcoxiboranos.

In other embodiments, the erasing agent is boran-N, N-diisopropylethylamine (BH3-DIPEA), boran-220 chloropyridine (BH3-CPy), boran-tetrahydrofuran (BH3-THF), or boran-dimethylsulfide (BH3-Me2S).

128

Scheme 10.

IMPI ^{, NSTI}? 7? Mexican OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0302.tif" />

Synthesis of a prodrug oligonucleotide precursor by chiral H-phosphonates (Route B)

<img file="MX342945B_D0303.tif" />

Fónrwla. one

<img file="MX342945B_D0304.tif" />

In another embodiment, described in Scheme 10, an achiral Hphosphonate of Formula 2 is treated with a condensation reagent to form an intermediate of structure II.

The structure II intermediate is not isolated and is treated in the same container with a chiral reagent to form a structure III chiral intermediate: The structure III intermediate is not isolated and is reacted in the same container with a nucleoside or nucleoside modified from structure IX to provide a chiral phosphite compound of structure X. In some embodiments, the structure

MEXICAN INSTITUTE ¿Λ

X is extracted in a solvent to separate it ~ ^{, J}

129 secondary, impurities and / or reagents. In other modalities, when the method is carried out by means of a solid phase synthesis, the solid support comprising the compound of structure X is filtered and separated from the secondary products, impurities and / or reagents. The compound of structure X is treated with an acid to remove the blocking group at the 5 'end of the growing nucleic acid chain (structure XI). The acidification step also removes the chiral auxiliary ligand to provide a chiral Hphosphonate IX. The 5 'unlocked intermediate is optionally allowed to re-enter the chain elongation cycle to form a fused intermediate containing a blocked 5' end which is then acidified to remove the 5 'end blocking group and the chiral auxiliary ligand.

When the desired chain length is reached, the 5 'deprotected intermediate is subjected to a modification step to introduce an X residue attached to each 2 0 of the phosphorous atoms to provide a compound of structure XII. The modified broker is unlocked by removing the remaining protecting groups, eg. , removing protecting groups from nucleobase, modified nucleobase, sugar or modified sugar, to provide a

130

ΙΜΡΙ

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0305.tif" />

Nucleic acid of Formula 1. In embodiments where the solid support is used, nucleic acid modified by a phosphorous atom is then cleaved from the solid support. In certain embodiments, the nucleic acid is left attached to the solid support for purification purposes and then cleaved from the solid support after purification. In one embodiment, the synthesis described in Scheme 10 is useful when the G1 and G2 positions of the chiral auxiliary ligand of Formula A are not hydrogen.

Modification of the compound of Formula IX obtained by Route B to introduce a residue of X- or X'phosphonate.

Other methods used to modify the compound of

Formula IX obtained by Route B are illustrated in

Reaction Schemes 10a and 10b. Phosphonate and phosphite are known to tautomerize and exist in equilibrium. The phosphite tautomer is less stable than the phosphonate tautomer. Equilibration takes place in the phosphonate tautomer under neutral conditions due to the strong P = O binding. Under acidic conditions, the phosphoryl group on the phosphonate becomes reversibly protonated. Cleavage of the PH junction in the intermediate occurs slowly to produce the phosphite intermediate. Structure IX is then modified to form structure XII, using reagents shown in

131 Reaction Schemes 10a and 10b. Reaction Scheme 10a.

IMPI • MEXICAN NSTITUTO OI THE INDUSTRIAL PROPERTY

<img file="MX342945B_D0306.tif" />

Modification of the phosphorus center of intermediates synthesized by Route B using an initial halogenation in phosphorus.

op, o o-vo

0a ^ΗΟγΥ '° -p- ° ^HOv ° pj R²

IX ° » ^H0UCLj^to | R²HO. 0

Oo .0. · Puxilive halogen reagent^H0~ P ° Y • A o

^x ° and ° P^to

P

Oa ^HOy ° P^to | R² %-⁰

RO 'Ό-ι, ο.

OR"

R² r ^H0Y ° P^to to go²0 ^ .0 rs' ~^orI j*

P

Oa

Reaction Scheme 10b.

Modification of phosphorus in intermediates synthesized by Route B using initial silylation.

<img file="MX342945B_D0307.tif" />

^H0T., O.,

HO. pl

IVHVTIVO of Nilil Ation

ROyo-j¹⁸

R² ηΆ

P

Agent tle i 'nciiarión p¡Si0. .or

I ^R2 l

0a

Η0- | Λ $ «

KP ° yM^to

P roil de »IquiÍMC¡ón electrofixing agent _k sulfur

RQyO'J³ or" ^RR ° yo.p

P

0a

Ηθη or Ba

Y 'Z HjB' ~ Όί, o ^ ^H° I ^^to

P²^θ* κ · ° i Se '“VO ¿A ^Η0γ ^⁸ ° T &lt;° zs^- '° yoJ^s

P

Oa

IMPif ~

132

MEXICAN INSTITUTE '

OF THE INDUSTRIAL MONEDAD

<img file="MX342945B_D0308.tif" />

This is done by reacting Structure IX with a halogenation reagent and then reacting it with a nucleophile. In specific embodiments, the halogenation reagent is CC14, CBr4, CI4,, C12, Br2, 12, sulfuryl chloride (SO2C12), phosgene, triphosgene, sulfur monochloride, sulfur dichloride, chloramine, CuC12, N-chlorosuccinimide (NCS), N-bromosuccinimide (NBS) or Nyodosuccinimide (NIS). In other specific embodiments, the halogenation reagent is CC14, CBr4, C12, sulfuryl chloride (SO2C12), or N-chlorosuccinimide (NCS). In some embodiments, the nucleophile is primary or secondary amines, alcohols, or thiols. In other embodiments, the nucleophile is NRfRfH, RfOH, or RfSH, where Rf is hydrogen, alkyl, alkenyl, alkynyl, or aryl, and at least one of Rf of NRfRfH is not hydrogen.

The modification step can also be performed by reacting structure IX with a silylation reagent, and then reacting it with a sulfur electrophile, a selenium electrophile, a biting agent, an alkylating agent, an aldehyde, or an acylating agent.

In specific embodiments, the silylation reagent is chlorotrimethylsilane (TMS-C1), triisopropylsilyl chloride (TIPS-C1), t-butyldimethylsilyl chloride

<img file="MX342945B_D0309.tif" />

₁₃₃ IMPI

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL (TBDMS-C1), t-butyldiphenylsilyl chloride (TBDPS-C1),

1,1,1,3,3,3-hexamethyldisilazane (HMDS), Ntrimethylsilyldimethylamine (TMSDMA), Ntrimethylsilyldiethylamine (TMSDEA), N-trimethylsilylacetamide (TMSA), N, O-bis (trimethylsilyl) acetamide (BSA), or N, 0bis (trimethylsilyl) trifluoroacetamide (BSTFA).

In other specific embodiments, the sulfur electrophile is a compound having one of the following formulas:

S8 (Formula B), Z10-SS-Z11 or Z10-SX-Z11, where Z10 and Zll are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide or thiocarbonyl or Z10 and Zll are taken together to form a 3- to 8-membered alicylic or heterocyclic ring, which may be substituted or unsubstituted; X is SO2, 0 or NRf; and Rf is hydrogen, alkyl, alkenyl, alkynyl, or aryl. In other embodiments, the sulfur electrophile is a compound of Formula B, C, D, E, or F:

<td>Sg</td><td>nh₂</td>

<td>Formula b</td><td>Formula c</td>

OEt

Formula θ

<img file="MX342945B_D0310.tif" />

OR

Formula E

<img file="MX342945B_D0311.tif" />

Formula E

IMPIOS,

134

OF THE

INDUSTRIAL

In other embodiments, the sulfur electrophile is

MEXICAN INSTITUTE

DELA FRRUPIEDAO *

Formula F, Formula E or Formula B.

In some embodiments, the selenium electrophile is a compound that has one of the following formulas:

Se (Formula G), Z10-Se-Se-Zll or Z10-Se-X-Zll, where Z10 and Zll are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide or thiocarbonyl or Z10 and Zll are taken together to form a 3-8 membered alicyclic or heterocyclic ring, which may be substituted or unsubstituted; X is SO2, S, 0 or NRf; and Rf is hydrogen, alkyl, alkenyl, alkynyl, or aryl.

In other modalities,

<td>compound</td><td>by Fómula</td><td>G, Η, I,</td>

<td>I know</td><td>KSeCN</td><td>I know Ph-P-Ph ¿H</td>

<td>Formula g</td><td>Formula 1</td><td>Formula i</td>

the selenium electrophile is a

J, K or L.

<img file="MX342945B_D0312.tif" />

Formula J Formula K Formula L

In some embodiments, the electrophile

Formula G or Formula L.

In some embodiments, the agent boran-N, N-diisopropylethylamine (BH3-DIPEA), from selenium is boronation, is boran-pyridine (BH3-Py), boran-2-chloropyridine (BH3-CPy), boran-aniline

135 (ΒΗ3 · Αη), boran-tetrahldrofuran dimethylsulfide (BH3-Me2S),

IMPI (BH3-THF)

MEXICAN INSTITUTE OF LA MONEDAD

INDUSTRIAL

<img file="MX342945B_D0313.tif" />

borananiline-cyanoborane, triphenylphosphine-carboalkoxyboranes. In other embodiments, the erasing agent is boran-N, N-diisopropylethylamine (BH3-DIPEA), boran-2-chloropyridine (BH3-CPy), borantetrahydrofuran (BH3-THF), or boran-dimethylsulfide (BH3-Me2S).

In other embodiments, the alkylating agent is an alkyl halide, alkenyl halide, alkynyl halide, alkyl sulfonate, alkenyl sulfonate, or alkynyl sulfonate.

In other embodiments, the aldehyde is (para) formaldehyde, alkyl aldehyde, alkenyl aldehyde, alkynyl aldehyde, or aryl aldehyde.

In still other embodiments, the acylating agent is a compound of Formula Μ or N:

G 'OG

Formula M Formula N where G7 is alkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy or heteroaryloxy; and M is F, Cl, Br, I, 3nitro-1,2,4-triazole, imidazole, alkyltriazole, tetrazole, pentafluorobenzene or 1-hydroxybenzotriazole.

<img file="MX342945B_D0314.tif" />

Scheme 11. Synthesis of a f osf orotideftS «5^R1AL

<img file="MX342945B_D0315.tif" />

136 chiral dinucleoside by stereoselec'ftvav ia synthesis

TBDPS

<img file="MX342945B_D0316.tif" />

Ph?

<img file="MX342945B_D0317.tif" />

ta

TBDPS

<img file="MX342945B_D0318.tif" />

^ NH CK ... i '

one. Ar-n

2.

<img file="MX342945B_D0319.tif" />

TBDPSO

3. DBU

<img file="MX342945B_D0320.tif" />

ta

TBDPSO

A method of synthesis of stereoselective dinucleoside phosphorothioate involves the use of stereochemically pure 3'-phosphoramidites, as described in Oka et al, (J. Am. Chem, Soc. (2003), 125: 8307-17). As shown in Scheme 6a (above), the 215 chloroxazaphospholidine derivatives are allowed to react with a 5'-0 (TBDPS) nucleoside to provide the 3'-0oxazaphospholidine derivative. Reaction of a 3'-O- (TBDPS) nucleoside with a 3'-Q-oxazaphospholidine derivative in the presence of an activator, such as N- (cyanomethyl) pyrrolidone, gives the phosphite dinucleoside as a simple diastereomer. The phosphite dinucleoside can be converted to phosphorothioate by a three-step process involving acetylation with acetic anhydride, sulfurization with Beaucage's reagent (3H-1,2-benzodithiol-3-one-1,1-dioxide; Iyer et al. , J. Am.

137

MEXICAN INSTITUTE

OF THE PROPERTY

Chem. Soc. (1990), 112: 1253-4), and cleavage of the chiral auxiliary ** with excess DBU. The phosphorothioate of the protected dinucleosis is then converted to the prodrug by methods described herein.

Other useful methods for the synthesis of dinucleoside phosphorothioates include enzymatic methods (Hacia et al. Biochemistry (1994), 33: 5367-9; Tang et al. Nucleosides

Nucleotides (1995), 14: 985-990), methods involving the separation of mixtures of diastereomeric phosphorothioate 10 prepared by non-stereoselective methods (Zon et al.

Oligonucleotides and Analogues: A Practical Approach; IRL

Press: London, 1991, pp 87-108) and methods involving stereoselective synthesis of phosphorothioates (Wilk et al. J.

Am. Chem. Soc. 2000, 122, 2149-2156; Lu et al, Angew. Chem.,

Int. Ed. 2000, 39, 4521-4524; Iyer et al

Tetrahedron.-Asymmetry 1995, 6, 1051-1054. Iyer et al

Tetrahedron Lett. 1998, 39, 2491-2494; Lu et al Tetrahedron

2001, 57, 1677-1687. Stec et al Nucleic Acids Res. 1991, 19,

5883-5888; Stec et al J. Am. Chem, Soc. 1995, 117, 1201920 12029; Uznan'ski et al J. Am. Chem. Soc. 1992, 114, 1019710202,

Reverse synthesis of nucleic acid 5 'to 3¹'

A nucleic acid of Formula 1 comprises a chiral Xphosphonate residue alternatively synthesized from the 5 'to 3' direction. In modalities in which a

138

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0321.tif" />

solid support, the nucleic acid binds to the solid support through its 5 'end of the growing nucleic acid, thereby presenting its 3' group for reaction, including enzymatic reaction (eg, ligation and polymerization). In some embodiments, this orientation is designed by preparing nucleoside monomers comprising an achiral Hphosphonate residue at the 5 'position and a protected hydroxyl group at the 3' position. In one embodiment, the nucleic acid is synthesized according to Scheme 12. In

Scheme 12, -R4 is -Orh, as defined above, the last cycle of synthesis, is R4, which is equivalent to Rl as defined herein.

Scheme 12.

Synthesis 5'to 3 'of a pro-oligonucleotide of Formula 1 comprising a chiral X-phosphonate residue.

n activation ίτ-η, · Ha / y ·&gt;

qurraF reagent Λ '

Ga ~ »r, β * v 1 condensation

<img file="MX342945B_D0322.tif" />

chain lengthening cycle acidify (3 'end release)

-ι na »

V remove other protecting groups, excise from solid support

ÓH Modification formula

Q.

<img file="MX342945B_D0323.tif" />

chiral auxiliary is removed during acidification

XV rí

<img file="MX342945B_D0324.tif" />

V

9

ΙΜΡΙ

<img file="MX342945B_D0325.tif" />

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL »In the embodiment described in Scheme 12, an achiral Hphosphonate of structure Ir is treated with a condensation reagent to form an intermediate of structure

Ilr. The Ilr structure intermediate is not isolated and is treated in the same container with a chiral reagent to form an Illr structure intermediate. The intermediate of structure Illr is not isolated and is reacted in the same container with a nucleoside or modified nucleoside of structure XIII to provide a chiral phosphite compound of structure XIV. In some embodiments, structure XIV is extracted in a solvent to separate it from secondary products, impurities, and / or reagents. In other embodiments, when the method is performed by solid phase synthesis, the solid support comprising the compound of structure XIV is filtered and separated from the by-products, impurities and / or reagents. Compound of structure XIV is treated with an acid to remove the blocking group at the 3 'end of the growing nucleic acid chain (structure XV). The acidification step also removes the chiral auxiliary ligand to provide a compound of structure XIII. The unlocked broker

3 'is optionally allowed to re-enter the chain lengthening cycle to form a condensed intermediate containing a blocked 3' end which is then

140

<img file="MX342945B_D0326.tif" />

Acidify to remove the 3 'end blocking group and chiral auxiliary ligand. After at least one cycle of chain lengthening, the 3 'unprotected intermediate undergoes a modification step to introduce a residue of

X attached to each of the phosphorous atoms to provide a compound of structure XVI. The modified broker is unlocked by removing the remaining protecting groups, eg. , removing protecting groups from nucleobase, modified nucleobase, sugar or modified sugar, to provide a nucleic acid of Formula 1. In other embodiments, the nucleoside comprising a 3'-OH moiety is an intermediate to a previous chain elongation cycle, as described herein. In still other embodiments, the nucleoside comprising a 3'-OH moiety is an intermediate obtained from another known synthetic nucleic acid method. Following a cycle of synthesis with the first nucleoside, nucleosides, nucleotides, or nucleic acids containing an unprotected -OH moiety can be used for subsequent elongation cycles. In embodiments where a solid support is used, the nucleic acid modified by a phosphorous atom is then cleaved from the solid support located at the 5 'end. In certain embodiments, nucleic acids are left attached to the solid support for purification purposes and are then cleaved from the

141

ΙΜΡΪ

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0327.tif" />

solid support after purification. In one aspect, the synthesis described in Scheme 12 is useful when the Gl and G2 positions of the chiral auxiliary ligand of Formula A are not hydrogen. 5 'in 3' reverse synthesis can be accomplished using the same starting materials in the Scheme in a mechanism analogous to the steps in Route A.

Creation of phosphotiotrieters with reversible protecting groups from H-phosphonate

Phosphorothioates can be stereospecifically synthesized from H-phosphonates with retention of configuration at the phosphorus atom (J. Org. Chem. 1991,

3861-3869). Also contemplated is the use of this reaction to synthesize phosphorothiotresters using a thiol-containing moiety that also carries a bioreversible protecting group; see Scheme 13. Additionally, stereocontrolled solid phase synthesis of oligonucleoside H-phosphonates has also been reported (Angew. Chem. Int. Ed. 2009, 48, 496-499) and it is contemplated that this method combined with alkylation during solid support synthesis, prepares phosphotiotriesters on the support.

142

Scheme 13

<img file="MX342945B_D0328.tif" />

O ü

DMTrO

<img file="MX342945B_D0329.tif" />

DMTrO

DMTrO HO

i) Ij.ACN: Py (3: 2), TBDPSC1 and the corresponding thiol ii) 3% DCA / DCM

Reaction conditions and reagents used in the methods of the invention.

Terms

Steps to react a molecule comprising an achiral H-phosphonate residue and a nucleoside comprising a 5'-OH residue to form a condensed intermediate can occur without isolating intermediates. In some embodiments, the steps for reacting a molecule comprising an achiral H-phosphonate residue and a nucleoside comprising a 5'-OH residue to form a condensed intermediate occurs in a one-pot reaction. In one embodiment, a molecule comprising an achiral H-phosphonate residue, condensation reagent, chiral agent, and a compound comprising a free nucleophilic residue are added to the reaction mixture at different times. In another embodiment, a molecule comprising an achiral H-phosphonate residue, reactive of

143

<img file="MX342945B_D0330.tif" />

IMPI

MEXICAN INSTITUTE

OF EA PROPERTY

INDUSTRIAL condensation and chiral reagent is present in the same reaction container or in the same container. In another embodiment, a molecule comprising an achiral H-phosphonate residue, condensation reagent, chiral agent, and a compound comprising a free nucleophilic residue are present in the same reaction or in the same container. This allows the reaction to proceed without isolation of intermediates and removes time consuming steps, resulting in economical and efficient synthesis. In specific embodiments, the achiral H-phosphonate, condensation reagent, chiral amino alcohol, 5'-OH nucleoside are present at the same time in a reaction. In a further embodiment, the formation of the chiral intermediate for condensation is formed in situ and is not isolated prior to the condensation reaction. In another embodiment, a molecule comprising an achiral H-phosphonate residue was activated by reaction with a condensation reagent, chiral reagent in a different reaction container than that used by reacting the chiral intermediate with the compound comprising a residue of 5¹-Free OH.

Synthesis on solid support

In some embodiments, nucleic acid syntheses are performed in solution. In other embodiments, nucleic acid syntheses are performed in the solid phase. The groups

144 —

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL solid support reagents can be unprotected or protected. During oligonucleotide synthesis, a solid support is treated with various reagents in different synthesis cycles to achieve step-by-step elongation of a growing oligonucleotide chain with individual nucleotide units. The nucleoside unit at the end of the chain that attaches directly to the solid support is called the first nucleoside, as used herein. The first nucleoside is attached to the solid support via a linker moiety, that is, a diradical with covalent linkages to the polymer of the solid support and the nucleoside. The bond remains intact during the synthesis cycles performed to assemble the oligonucleotide chain and is cleaved after assembly of the chain to release the oligonucleotide from the support.

Solid supports for solid phase nucleic acid synthesis include supports described herein, eg. , US Patents 4,659,774, 5,141,813,

4,458,066; Caruthers, US Patent No. 4,415,732,

4,458,066, 4,500,707, 4,668,777, 4,973,679 and 5,132,418;

Andrus et al ,. U.S. Patent No. 5,047,524,

5,262,530; and Koster, US Patent No. 4,725,677 (reissued as Re34,069). In some embodiments, the solid phase is an organic polymeric support. In others

IΜ ΡI

MEXICAN INSTITUTE '¿íínZ

OF THE PKOPIÍaII V support $ ÓÍ^Ti'frtérr &amp; § ^ support polllllél ico -

145 üoliestereno, a modalities, the solid phase is an inorganic. In some embodiments, the organic is polystyrene, polyethylene glycol-polystyrene graft aminomethyl copolymer, polyacrylamide, polymethacrylate, polyvinyl alcohol, highly crosslinked polymer (HCP), or other synthetic polymers, carbohydrates such as cellulose and starch, or other polymeric carbohydrates or other polymers. copolymer, composites, or combinations of the above inorganic or organic materials. In other embodiments, the inorganic polymeric support on silica, aluminum, controlled polyglass (CPG) which is a support for silica gel or aminopropyl CPG. Other useful solid supports include fluorine solid supports (see eg.

WO / 2005/070859), Long Chain Alkylamine (LCAA) Solid Controlled Glass Pore (CPG) Supports, (see eg, SP Adams, KS Kavka, EJ Wykes, SB

Holder and GR Galluppi, J. Am. Chem. Soc., 1983, 105, 661663; GR Gough, MJ Bruden and Ρ. T. Gilham, Tetrahedron

Lett., 1981, 22, 4177-4180). Membrane supports and polymeric membranes (see, eg, Innovation and Perspectives in

Solid Phase Synthesis, Peptides, Proteins and Nucleic Acids, ch 21 pp 157-162, 1994, Ed. Roger Epton and US Patent No. 4,923,901) are also useful for the

145 nucleic acid synthesis. Once formed, a membrane

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0331.tif" />

it may be chemically functionalized for use in nucleic acid synthesis. In addition to binding a functional group to the membrane, the use of a membrane-bound linker or spacer group can be used to minimize the spherical obstacle between the membrane and the synthesized chain.

Other suitable solid supports include those generally known in the art as suitable for use in solid phase methodologies, including, for example, glass sold as PrimerTM 200 support, controlled glass pore (CPG), oxalyl controlled glass pore ( see, eg, Alul, et al., Nucleic Acids Research, 1991,

19, 1527), TentaGel Support, a derivatized aminopolyethylene glycol support (see, eg, Wright, et al.,

Tetrahedron Lett., 1993, 34, 3373), and Poros, a polystyrene / divinylbenzene copolymer.

The use of surface activated polymers in the synthesis of natural and modified nucleic acids and proteins in various solid support media has been demonstrated.

The solid support material can be any adequately uniform polymer in porosity, has sufficient amine content and is flexible enough to undergo any auxiliary manipulation without losing

<img file="MX342945B_D0332.tif" />

147 ΙΜΡΙ

MEXICAN INSTITUTE

OF THE PROPERTY . INDUSTRIAL 7 * —I integrity. Examples of suitable selected polypropylene materials, "~~ include nylon, polytetrafluoroethylene, polyester, polystyrene, polycarbonate, and nitrocellulose. Other materials can serve as the solid support depending on the researcher's design. In consideration of some designs, for example, a coated metal, in particular gold or platinum, can be selected (see, eg, US Publication No. 20010055761).

In an oligonucleotide synthesis embodiment, for example, a nucleoside is anchored to a solid support that is functionalized with hydroxyl or amino residues. Alternatively, the solid support is derivatized to provide an acid labile trialkoxytrityl group, such as a trimethoxytryl (TMT) group. Without being bound by theory, it is expected that the presence of the trialkoxytrityl protecting group will allow initial detritylation under conditions commonly used in DNA synthesizers. For faster release of the oligonucleotide material in solution with aqueous ammonia, a diglycol bond is optionally inserted into the support.

Rest of link

A binding or linking moiety is optionally used to connect the solid support to the compound comprising a free nucleophilic moiety. Such suitable links are known

148

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY as short molecules that serve to connect a solid support with the functional groups (eg, hydroxyl groups) of initial nucleoside molecules in solid phase synthesis techniques. In some embodiments, the bond moiety is a succinamic acid bond or a succinate bond (-C0CH2-CH2-C0-) or an oxalyl bond (-CO-CO-). In other embodiments, the linker moiety and nucleosides are linked together through an ester linkage. In other embodiments, the binding moiety and nucleosides are linked together through an amide bond. In additional embodiments, the linker moiety connects the nucleoside to another nucleotide or nucleic acid. Suitable links are described in, for example,

Oligonucleotides And Analogues A Practical Approach, Ekstein,

F. Ed., IRL Press, NY, 1991, Chapter 1.

A linking moiety is used to connect the compound comprising a free nucleophilic moiety to another nucleoside, nucleotide, or nucleic acid. In some embodiments, the bond moiety is a phosphodiester bond.

In other embodiments, the linker moiety is an Hphosphonate moiety. In still other embodiments, the linker moiety is an H-phosphonate moiety.

Solvents for synthesis

The synthesis of nucleic acids is carried out in an aprotic organic solvent. In some modalities, the

9

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0333.tif" />

solvent is acetonitrile, pyridine, tetrahydrofuran, or dichloromethane. In some embodiments, when the aprotic organic solvent is not basic, a base is present in the reaction step. In some embodiments, in which a base is present, the base is a pyridine, quinoline, or N, N-dimethylaniline. Other examples of bases include pyrrolidine, piperidine, N-methyl pyrrolidine, pyridine, quinoline, N, N-dimethylaminopyridine (DMAP), or N, N-dimethylaniline. In some embodiments, the aprotic organic solvent is anhydrous. In other embodiments, the anhydrous aprotic organic solvent is freshly distilled. In some embodiments, the freshly distilled anhydrous aprotic organic solvent is pyridine. In other embodiments, the freshly distilled anhydrous aprotic organic solvent is tetrahydrofuran. In other embodiments, the freshly distilled anhydrous aprotic organic solvent is acetonitrile.

Acidification conditions to remove blocking groups.

Acidification to remove blocking groups is accomplished by Bransted acid or Lewis acid. In some embodiments, acidification is used to remove Rl-blocking groups. Useful Br0nsted acids are carboxylic acids, alkylsulfonic acids, arylsulfonic acids, phosphoric acid and its derivatives, phosphonic acid and its

150

<img file="MX342945B_D0334.tif" />

derivatives, alkylphosphonic acids and their derivatives, arylphosphonic acids and their derivatives, phosphonic acid, dialkylphosphonic acids and diarylphosphinic acids having a pKa value (25 ° C in water) of -0.6 (trifluoroacetic acid) in 4.76 (acetic acid) in a organic solvent or water (in the case of 80% acetic acid). The acid concentration (1 to 80%) used in the acidification step depends on the acidity of the acid. The strength of the acid should be taken into account as strong acid conditions will result in purification / depirimidination, where the purinyl or pyrimidyl bases are cleaved from the ribose ring.

RCOOH

RS-OH

Ik

R¹OI ^ -OH

ÓR² or

R¹- ^ - OH

ÓR²

R¹-f ^ -OH

R²

R = H, alkyl, aryl R = alkyl, aryl R¹, R² = H. Alkyl, aryl R¹, R² = H, alkyl, aryl R¹, R² = H. Alkyl, aryl

In some embodiments, acidification is accomplished by Lewis acid in an organic solvent. Lewis acids are ZnX2 where X is Cl, Br, I, or CF3SO3.

In some embodiments, acidification involves adding an amount of Bronsted or Lewis acid effective to convert the condensed intermediate to the compound of

Formula 4 without removing the purine residues from the condensed intermediate.

Acids that are useful in the acidification step also include, but are not limited to, 10% phosphoric acid.

151

IMPI

MEXICAN INSTITUTE OF INOUSTRJAL PROPERTY

<img file="MX342945B_D0335.tif" />

in an organic solvent, 10% hydrochloric acid in an organic solvent, 1% trifluoroacetic acid in an organic solvent, 3% dichloroacetic acid in an organic solvent, or 80% acetic acid in water. The concentration of any Bronsted or Lewis acid used in the process is selected such that the concentration of the acid does not exceed a concentration that causes cleavage of the nucleobase from the sugar moiety.

In some embodiments, acidification involves adding 1% trifluoroacetic acid in an organic solvent.

In some embodiments, acidification involves adding about 0.1% to about 8% trifluoroacetic acid in an organic solvent. In other embodiments, acidification involves adding 3% dichloroacetic acid in an organic solvent. In other embodiments, acidification involves adding about 0.1% to about 10% dichloroacetic acid in an organic solvent. In still other modalities, acidification involves adding 3% trichloroacetic acid in an organic solvent. In still other embodiments, acidification involves adding about 0.1% to about 10% trichloroacetic acid in an organic solvent. In some embodiments, acidification involves adding 80% acetic acid to water. In some modalities, acidification

152

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0336.tif" />

It comprises adding approximately 50% to approximately 90%, or approximately 50% to approximately 80%, approximately 50% to approximately 70%, approximately 50% to approximately 60%, approximately 70% to approximately 90% acetic acid in water. In some embodiments, acidification further comprises adding cation binders to the acidic solvent. In specific embodiments, the cation binders can be triethylsilane or triisopropylsilane. In some embodiments, Rl is released prior to the acidification step of the condensed intermediate. In some embodiments, Rl is unlocked by acidification which involves adding 1% trifluoroacetic acid in an organic solvent. In some embodiments, Rl is unlocked by acidification which involves adding 3% dichloroacetic acid in an organic solvent. In some embodiments, Rl is unlocked by acidification which involves adding 3% trichloroacetic acid in an organic solvent.

Suppression of the remains or blocking groups

Functional groups such as hydroxyl or amino residues that are located on nucleobases or sugar residues are routinely blocked with blocking (protecting) groups (residues) during synthesis and subsequently unblocked.

In general, a blocking group provides chemical functionality of an inert molecule under reaction conditions

IMPI - * ζ

MEXICAN INSTITUTE

R 7 OF PROPERTY V&gt; * M

INDUSTRIAL specific and can then be removed from such functionality in a molecule without substantially damaging the rest of the molecule (see eg, Green and Wuts, Protective Groups in

Organic Synthesis, 2^to Ed., John Wiley &amp; Sons, New York,

1991). For example, amino groups can be blocked with nitrogen blocking groups such as phthalimido, 9fludrenylmethoxycarbonyl (FMOC), triphenylmethylsulfenyl, tBOC, 4,4'-dimethoxytrityl (DMTr), 4-methoxytryl (MMTr), 9phenylxanthine-9-yl ( Pixyl), triphenyl (Tr) or 9- (p10 methoxyphenyl) xanthine-9-yl (MOX). Carboxyl groups can be protected as acetyl groups. Hydroxyl groups can be protected such as tetrahydropyranyl (THP), tbutyldimethylsilyl (TBDMS), 1- [(2-chloro-4-methyl) phenyl] -4methoxypiperidin-4-yl (Ctmp), 1- (2-fluorophenyl) - 415 methoxypiperidin-4-yl (Fpmp), 1- (2-chloroethoxy) ethyl, 3methoxy-1,5-dicarbomethoxypentan-3-yl (MDP), bis (2acetoxyethoxy) methyl (ACE), triisopropylsilyloxymethyl (TOM),

1- (2-cyanoethoxy) ethyl (CEE), 2-cyanoethoxymethyl (CEM), [4- (N-dichloroacetyl-N-methylamino) benzyloxy] methyl, 2-cyanoethyl (CN), pivaloyloxymethyl (PivOM), levunyloxymethyl (ALE). Other representatives of hydroxyl blocking groups have been described (see, eg, Beaucage et al., Tetrahedron, 1992,

46, 2223). In some embodiments, the hydroxyl blocking groups are acid labile groups, such as triphyl,

154

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MEXICAN INSTITUTE

OF PROPERTY J

INDUSTRIAL

<img file="MX342945B_D0337.tif" />

monomethoxytryl, dimethoxytryl, trimethoxytryl, phenylxanthine-9-yl (Pixyl), and 9- (p-methoxyphenyl) xanthine-9-yl (MOX). Chemical functional groups can also be blocked including as a precursor. Therefore, an azido group can be considered a blocked form of an amine as the azide group is readily converted to the amine. Representatives of additional protecting groups used in nucleic acid synthesis are known (see, eg, Agrawal et al., Protocols for Oligonucleotide

Conjugates, Eds., Humana Press, New Jersey, 1994, Vol. 26, pp. 1-72).

Different methods are known and are used for the suppression of nucleic acid blocking groups. In some embodiments, all blocking groups are removed.

In other embodiments, the blocking groups are partially removed. In still other embodiments, the reaction conditions can be adjusted to remove the blocking groups on certain residues. In some embodiments where R2 is a blocking group, the deletion of the blocking group 20 in R2 is orthogonal to the deletion of the blocking group in Rl. The blocking groups in Rl and R2 remain intact during the synthesis steps and are collectively removed after chain assembly. In some embodiments, R2 blocking groups are removed

IM --- 155

INSTITUTC OF THE

<img file="MX342945B_D0338.tif" />

simultaneously with cleavage of the nucleic acids from the solid support and with the deletion of the nucleobase blocking groups. In specific embodiments, the blocking group in Rl is removed while the blocking groups in R2 and the nucleobases remain intact. The Rl blocking groups are cleaved on solid supports with an organic base such as a primary amine, a secondary amine, or a mixture thereof. Unlocking the Rl position is commonly referred to as frontal unprotection.

In one embodiment, the nucleobase blocking groups, if any, are cleaved after assembly of the corresponding nucleic acid with an acidic reagent. In another embodiment, one or more of the nucleobase blocking groups are cleaved under conditions that are neither acidic nor basic, eg. , cleavable with hydrofluoric acid complexes or fluoride salts. In yet another embodiment, one or more nucleobase blocking groups are cleavable after assembly of the corresponding nucleic acid in the presence of a base or basic solvent and where the nucleobase blocking group is stable under the conditions of the frontal amine deprotection step.

In some embodiments, nucleobase blocking groups are not required. In other embodiments, nucleobase blocking groups are required. In still others

156 modalities, certain nucleobases need a group

IMPI

MEXICAN INSTITUTE D (INDUSTRIAL PROPERTY

<img file="MX342945B_D0339.tif" />

blocking while other nucleobases do not need blocking groups. In embodiments where the nucleobases are blocked, the blocking groups are removed in whole or in part under appropriate conditions to remove the blocking group at the front end. For example, Rl can denote ORa, where Ra is acyl and Ba denotes guanine blocked with an acyl group which includes, but is not limited to, isobutyryl, acetyl or 4 - (tert-butylphenoxy) acetyl. The acyl groups in Rl and Ba will be deleted or partially deleted during the same unlocking step.

Reagents

Condensation reagent

The condensation reagent (CR) useful in the methods of the invention has one of the following general formulas:

—L,

Cr4

TO

I

Z *

C_h5 cycloalkyl, where Zl, Z2, Z3, Z4, Z5, Z6, Z7, Z8 and Z9 are independently selected from alkyl, aminoalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy or heteroaryloxy, or where any of Z2 and Z3, Z5 and Z6, Z7 and ₁₅₇ IMPI 5? * Mexican institute

OF THE PROPERTY

INDUSTRIAL

Z8, Z8 and Z9, Z9 and Z7, or Z7 and Z8 and Z9 are taken together to form a 3- to 20-membered alicyclic or heterocyclic ring; Q- is a counter anion; and L is an outgoing group.

In some embodiments, the counter ion of the CR condensation reagent is Cl-, Br-, BF4-, PF6-, TfO-, Tf2N-, AsF6-,

C1O4- or SbF6-, where Tf is CF3SO2. In some embodiments, the leaving group of the condensation reagent CR is F, Cl, Br,

I, 3-nitro-l, 2,4-triazole, imidazole, alkyltriazole, tetrazole, pentafluorobenzene or 1-hydroxybenzotriazole.

Examples of condensing agents that can be used in the process include, but are not limited to, pentafluorobenzoyl chloride, carbonyldiimidazole (CDI), 1-methylenesulfonyl-3-nitrotriazole (MSNT), 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide hydrochloride (EDCI-HCl), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (PyBOP), N, N'bis (2-oxo-3-oxazolidinyl) phosphinicq (BopCl) chloride, 2- (ΙΗ-7-azabenzotriazol-l-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and O-benzotriazole-Ν, Ν, N ', N'20 tetramethyluronium (HBTU) hexafluorophosphate. DIPCDI; N, N'-bis (2-oxo-3oxazolidinyl) phosphonic bromide (BopBr), 1,3-dimethyl-2- (3-nitro-l, 2,4-triazol-1-yl) -2-pyrrolidin- hexafluorophosphate l-yl1,3,2-diazaphospholidinium (MNTP), 3-nitro1,2,4-triazol-1-yl-tris (pyrrolidin-1-yl) phosphonium (PyNTP) hexafluorophosphate,

158

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MEXICAN INSTITUTE PE INDUSTRIAL PROPERTY

<img file="MX342945B_D0340.tif" />

bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP);

Q- (benzotriazol-l-yl) -Ν, Ν, Ν ', N' (TBTU); and tetrafluoroborate tetramethyluronium tetramethylfluoroformamidinium hexafluorophosphate (TFFH).

In certain embodiments, the counter ion of the condensation reagent CR is

Cl-, Br-, BF4-, PF6-, TfO-, Tf2N-, AsF5-, C1O4- O SbF6-, where Tf is CF3SO2.

In other embodiments of the invention, the condensation reagent is 1- (2,4,6-triisopropylbenzenesulfonyl) -510 (pyridin-2-yl) tetrazolide, pivaloyl chloride, bromotrispyrrolidinophosphonium hexafluorophosphate,

N, N'-bis (2-oxo-3-oxazolidinyl) phosphonic (BopCl) or 2-chloro5,5-dimethyl-2-oxo-1,3,2-dioxafosfinan. In one embodiment, the condensation reagent is N, N'-bis (2-oxo-315 oxazolidinyl) phosphinic chloride (BopCl). Other known condensation reagents have been described (see, eg,

WO / 2006/066260).

In other embodiments, the condensation reagent is 1,3-dimethyl-2- (3-nitro-1,2,4-triazol-120 yl) -2-pyrrolidin-l-yl-l, 3,2- hexafluorophosphate. diazaphospholidinium (MNTP) or 3-nitro-l, 2,4-triazol-l-yltris (pyrrolidin-l-yl) phosphonium (PyNTP) hexafluorophosphate.

159 to

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MtXICAN'j INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0341.tif" />

ΓΛ? ΓΛ w

BopCI / O PFe rV^{+ N}^kN^Z V \ \ C_n^ no!

MNTP • Pfr

V * TS

OR

PyNTP

N0₂

Chiral reagent

In the methods of the present invention, chiral reagents are used to confer stereoselectivity in the production of X-phosphonate bonds. Several different chiral auxiliaries can be used in this process which are compounds of Formula 3-1 where W1 and W2 are any of -0, -S- or -NG5-, which are capable of reacting with the H-phosphonate starting material, a compound of Formula 2 to form the chiral intermediate, as shown in the structure

III of Schemes 5 and 5.

H — VVj W₂-H

G ^ fuJu³'^¹

G³ G²

Formula 3-1

0 Ul and U3 are atoms if there were, or with each other if r double or triple. U2 is -C-,

-S- where r is a number heteroatoms are contiguous

<td>carbon that</td><td>I know</td><td>bind U2 if</td>

<td>is 0, by</td><td>a</td><td>simple union,</td>

<td>-CG8-, -CG8G8-,</td><td colspan="2">-NG8-, -N-, -O- 0</td>

<td>integer from 0 to 5</td><td>and</td><td>no more than two</td>

When any of U2 is C, it

<img file="MX342945B_D0342.tif" />

ΙΜΡΙ

0 MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL must form a triple union between a second instance of U2, which is C, or with one of UI or U3. Similarly, when any of U2 is CG8, a double junction must be formed between a second instance of U2 that is -CG8- or -N- or with one of

UI or U3.

For example, in some embodiments, -Ul- (U2) r-U3- is CG3G4-CG1G2-. In some modes, -Ul- (U2) r-U3 - is

CG3 = CG1-. In some embodiments, -Ul- (U2) r-U3 - is -C == C-. In some embodiments, -Ul- (U2) r-U3- is -CG3 = C G8-CG1G2-. In some embodiments, -Ul- (U2) r-U3- is -CG3G4-O-CG1G2-. In some embodiments, -Ul- (U2) r-U3- is -CG3G4-NG8-CG1G2-. In some embodiments, -Ul- (U2) r-U3- is -CG3G4-N-CG2-. In some embodiments, -Ul- (U2) r-U3- is -CG3G4-N = C G8-CG1G2-.

Gl, G2, G3, G4, G5 and G8 are independently hydrogen, alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl or aryl, or two of Gl, G2, G3, G4 and G5 are G6 taken together to form a ring that it contains a saturated, partially unsaturated or unsaturated heteroatom or carbocyclic of up to about 20-20 ring atoms that is monocyclic or polycyclic, and is fused or unfused. In some embodiments, the ring thus formed is replaced by oxo, thioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl residues. In some embodiments, when the ring formed by taking two₁₆₁ ΙΜΡΙΥ ^ γ Mexican Institute r ^ **** E¿- * e * J

DE LA PROREDAO CSw »aS * Ls £ &amp;

INDUSTRIAL

Together G6 is substituted, is substituted by a moiety that is bulky enough to confer stereoselectivity during the reaction.

For example, in some embodiments, the ring forming by taking two of G6 together is cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl, cyclopentenyl, tetrahydropyranyl, or piperazinyl.

In some embodiments of the invention, the chiral reagent is a compound of Formula 3.

H — Wi W₂-H

G³ G²

Formula 3

In some embodiments of Formula 3, W1 and W2 are independently -NG5-, -0- or -S-; Gl, G2, G3, G4 and G5 are independently hydrogen, alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclic, heteroaryl or aryl, or two of Gl, G2, G3, G4 and G5 are taken together to form a ring containing a heteroatom or carbocyclic saturated, partially unsaturated or unsaturated of up to about 20 ring atoms that is monocyclic or polycyclic, fused or non-fused and no more than four of Gl, G2, G3, G4 and G5 are G6. Similar to compounds of Formula 3 ', any of Gl, G2, G3, G4 or

G5 is replaced by oxo, thioxo, alkyl residues,

162

ΙΜΡΙ

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0343.tif" />

alkenyl, alkynyl, heteroaryl or aryl. In some modalities, said substitution induces stereoselectivity in the production of X-phosphonate.

In some embodiments of the invention, the chiral reagent has one of the following Formulas:

<td>g⁵-nh oh c</td><td>i⁵-NH SH</td><td>g⁵-nh hn-g °</td><td>HS OH</td><td>HO OH</td><td>HS SH</td>

<td>gCw</td><td> \ / _G4 * O'G¹</td><td></td><td>G⁴* W? G¹</td><td>G⁴yí; G¹</td><td>Q4; | '/ Q1</td>

<td>G³ G²</td><td>G³ G²</td><td>G³ G²</td><td>g³ g²</td><td>G³ G²</td><td>G³ G²</td>

<td>Formulas</td><td>3-A 3-B</td><td>3-C</td><td>3-D</td><td>3-E</td><td>3-F</td>

<td>In</td><td>some</td><td>modalities,</td><td colspan="3">the chiral reagent is a</td>

<td colspan="2">aminoalcohol. In</td><td colspan="2">some modalities, the</td><td>reagent</td><td>chiral is</td>

an aminothiol. In still other embodiments, the chiral reagent is an aminophenol. In some embodiments, the chiral reagent is (S) - and (R) -2-methylamino-1-phenylethanol, (IR, 2S) -ephedrine, or (IR, 2S) -2-methylamino-l, 2-diphenylethanol.

In other embodiments of the invention, the chiral reagent is a compound of one of the following Formulas:

<img file="MX342945B_D0344.tif" />

Formula 0 Formula P Formula Q Formula R

The choice of chiral reagent, for example, the isomer represented by Formula O or its stereoisomer, Formula P, allows specific control of chirality in phosphorus.

Therefore, an RP or SP configuration can be selected in each synthesis cycle, allowing control of the

163

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROHEDAÜ

<img file="MX342945B_D0345.tif" />

entirety of the three-dimensional structure of the nucleic acid product. In some embodiments of the invention, a nucleic acid product has all RP stereocenters. In some embodiments of the invention, a nucleic acid product has all the SP stereo centers. In some embodiments, the RP and SP centers are screened to provide a three-dimensional superstructure specific to the nucleic acid chain.

Stereochemistry of oligonucleoside phosphorothioate linkages

<td>The</td><td>oligonucleosides</td><td>phosphorothioates</td><td colspan="2">have shown</td>

<td>potential</td><td colspan="2">therapeutic (Stein et al.,</td><td>Science</td><td> (1993),</td>

<td> 261:1004-</td><td>12; Agrawal et al.</td><td>, Antisence Res.</td><td>and Dev.</td><td> (1992),</td>

<td> 2:261-66;</td><td>Bayever et al.,</td><td>Antisense Res.</td><td>and Dev.</td><td> (1993),</td>

3: 383-390). Phosphorothioate oligonucleosides prepared without regard to phosphorothioate stereochemistry exist as a mixture of 2n diastereomers, where n is the number of phosphorothioate internucleotide linkages. The chemical and biological properties of these diastereomeric phosphorothioates can be well differentiated. For example, Wada et al (Nucleic Acids Symposium Series No. 51 p. 119-120; doi: 10.1093 / nass / nrm060) found that the duplex - (Rp) (Ups) 9U / (Ap) 9A stereodefined exhibited a higher value of Tm than the natural - (Up) 9U / (Ap) 9A and the - ( Sp) - (Ups) 9U

IMPI

164

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL stereodefined did not form a duplex. In another example, in a study by Tang et al., (Nucleosides Nucleotides (1995), 14: 985-990), it was found that Rpoligodeoxyribonucleoside phosphorothioates have a lower stability with respect to nucleases that are endogenous to human serum than oligodeoxyribonucleoside phosphorothioates. major with undefined phosphorus chirality

Nucleobases and modified nucleobases

The Ba nucleobase of Formula 1 is a natural nucleobase or a modified nucleobase derived from natural nucleobases. Examples include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine, the corresponding amino groups of which are protected by protecting groups acyl, 2-fluorouracil, 2-fluorocytosine, 515 bromouracil, 5-iodouracil, 2,6-diaminopurine. , azacytosine, pyrimidine analogs, such as, pseudoisocytosine and pseudouracil, and other modified nucleobases, such as, 8-substituted purines, xanthine or hypoxanthine (the last two are the products of natural degradation). The modified nucleobases 20 described in Chiu and Rana, RNA, 2003, 9, 10341048, Limbach et al. Nucleic Acids Research, 1994, 22, 21832196 and Revankar and Rao, Comprehensive Natural Products

Chemistry, vol. 7, 313, are also considered to be Ba residues of Formula 1.

IMPI

MEXICAN INSTITUTE OF THE FROFIEOAI) INDUSTRIAL

Compounds represented by the general formulas below are also considered modified nucleobases:

<img file="MX342945B_D0346.tif" />

In the formulas above, R8 is a linear or branched alkyl, ariio, araiquyl or aryloxy alkyl group having 1 to 15 carbon atoms, including, by way of example only, a methyl, isopropyl, phenyl, benzyl or phenoxymethyl; and R9 and RIO each represent a linear or branched alkyl group having 1 to 4 carbon atoms.

Modified nucleobases also include size-expanded nucleobases to which one or more benzene rings have been added. The nucleic base substitution described in the Glen Research catalog (www.glenresearch.com); Krueger AT et al, Acc. Chem. Res., 2007, 40, 141-150; Kool,

ET, Acc. Chem. Res., 2002, 35, 936-943; Benner SA, et al. ,

Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, FE, et al. ,

Curr. Opin. Chem. Biol-, 2003, 7, 723-733; Hirao, I

Curr.

6 «MEXICAN NSTITUTE

OF PROPERTY \&gt; w »eaá3U @ F

INDUSTRIAL

Opin. Chem. Biol., 2006, 10, 622-627, are considered useful for the synthesis of the nucleic acids described herein. Here are some examples of these expanded size nucleobases:

<img file="MX342945B_D0347.tif" />

Herein, modified nucleobases also include structures that are not considered nucleobases but are other residues, such as, non-exhaustively, rings derived from corrin or porphyrin. Porphyrin-derived base substitutions are described in MoralesRojas, H and Kool, ET, Org. Lett., 2002, 4, 4377-4380. The following is an example of a ring derived from porphyrin that can be used as a base replacement:

<img file="MX342945B_D0348.tif" />

167

Other substitutions below:

modified base nucleobases, such as those that also include

IMPI

MEXICAN INSTITUTE OE LA INDUSTRIAL MOHEDAL

<img file="MX342945B_D0349.tif" />

are provided to

<img file="MX342945B_D0350.tif" />

LíWV

<img file="MX342945B_D0351.tif" />

<img file="MX342945B_D0352.tif" />

Modified nucleobases that are fluorescent are also considered. Non-limiting examples of these base substitutions include phenanthrene, pyrene, stilbene, isoxanthine, isozantopterin, terphenyl, tertiophene, benzotertiofen, coumarin, lumazine, linked stilbene, benzo-uracil, and naphtho-uracil, as shown below:

<img file="MX342945B_D0353.tif" />

168

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0354.tif" />

Modified nucleobases may be unsubstituted or contain additional substitutions, such as, heteroatoms, alkyl groups, or linker moieties connected to fluorescent moieties, biotin or avidin moieties, or other proteins or peptides. Modified nucleobases also include certain universal bases that are not nucleobases, in the traditional sense, but function similarly to nucleobases. A representative example of such a universal base is 3-nitropyrrole.

In addition to nucleosides of structure IV or IX, other nucleosides may also be used in the process described herein, including nucleosides incorporating modified nucleobases or nucleobases covalently linked to modified sugars. Some examples of nucleosides containing modified nucleobases include 4acetylcytidine; 5- (carboxyhydroxylmethyl) uridine; 2'-0methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine; 5carboxymethylaminomethyluridine; dihydride idina; 2'-0methylpseudouridine; beta, D-galactosylqueosine; 2'-0methylguanosine; N6-isopentenyladenosine; l-methyladenosine; 1methylpseudouridine; 1-methylguanosine; 1-methylinosine; 2,2dimethylguanosine; 2-methyladenosine; 2-methylguanosine; N7methylguanosine; 3-methyl-cytidine; 5-methylcytidine; N6methyladenosine; 7-methylguanosine; 5-methylaminoethyluridine; 5169

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0355.tif" />

methoxyamomethyl-2-thiouridine; beta, D-mannosylcheosine; 5methoxycarbonylmethyluridine; 5-methoxyuridine; 2-methylthio-N6-isopentenyladenosine; N - ((9-beta, D-ribofuranosyl-2-methylthiopurine-6-yl) carbamoyl) threonine; N - ((9-beta, Dribofuranosylpurine-6-yl) -N-methylcarbamoyl) threonine;

uridine-5-oxyacetic acid methyl ester; uridine-5-oxyacetic acid (v); pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine; 2-thiouridine; 4-thiouridine; 5-methyluridine; 2'-O-methyl-5-methyluridine and 2'-0-methyluridine.

In some embodiments, nucleosides include positionally modified bicyclic nucleoside analogs.

<td> 6¹ that have</td><td>a</td><td>chirality</td>

<td>include</td><td>the</td><td>analogues</td>

<td>American</td><td>No.</td><td> 7,399,845</td>

(R) or (S) in the 6 'position described in the Patent

In other embodiments, nucleosides include the 5'-position modified bicyclic nucleoside analogs that have a (R) or (S) chirality at the 5 'position and include the analogs described in US Patent Application Publication No.

20070287831.

In some embodiments, the nucleobases or modified nucleobases comprise biomolecule binding residues, such as, antibodies, antibody fragments, biotin, avidin, streptavidin, receptor ligands, or chelating residues. In other modes, Ba is 5170

ΙΜΡΙ

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

<img file="MX342945B_D0356.tif" />

bromouracil, 5-iodouracil or 2,6-diaminopurine. In other embodiments, Ba is modified by substitution for a fluorescent or biomolecule binding moiety. In some embodiments, the substituent on Ba is a fluorescent moiety. In other embodiments, the substituent on Ba is biotin or avidin.

Nucleotide / nucleoside modified sugars.

The most common naturally occurring nucleotides are ribose sugars attached to the nucleobases adenosine (A), cytosine (C), guanine (G), and thymine (T) or uracil (U). Modified nucleotides are also considered where the phosphate group or residues of modified phosphorous atoms in the nucleotides can bind to various positions of the sugar or modified sugar. As non-limiting examples, the phosphate group or the residue of the modified phosphorus atom can be attached to the 2 ', 3', 4 'or 5' hydroxyl residue of a sugar or modified sugar. Nucleotides containing the modified nucleobases described above can also be used in the process described herein. In some embodiments, nucleotides or modified nucleotides comprising an unprotected OH moiety are used in the process described herein.

In addition to the ribose moiety described in Schemes 14b, other modified sugars may also be incorporated. ₁₇₁ IMPI ^^{Χ Χ} MEXICAN INSTITUTE &amp; ***

OF THE PROPERTY

INDUSTRIAL in the nucleic acids described herein. In some embodiments, the modified sugars contain one or more substituents at the 2 'position, including one of the following: F; CF3, CN, N3, NO, N02, O-, S- or N-alkyl; OR-,

S- or N-alkenyl; 0-, S- or N-alkynyl; or 0-alkyl-0-alkyl, O-alkyl-N-alkyl, or N-alkyl-O-alkyl where the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C 1 -C 10 -alkyl, and alkynyl. Examples of substitutes include, but are not limited to, O (CH2) nOCH3 and

O (CH2) nNH2, where n is from 1 to about 10, MOE, DMAOE,

DMAEOE. Also considered herein are the modified sugars described in WO 2001/088198 and Martin et al. ,

Helv. Chim. Acta, 1995, 78, 486-504. In some embodiments, the modified sugars comprise substituted silyl groups, an RNA cleavage group, a reporter group, a fluorescent tag, an intercalator, a group to enhance the pharmacokinetic properties of a nucleic acid, or a group to enhance the pharmacodynamic properties of nucleic acid and other substitutes with similar properties. Modifications may be made at the 2 ', 3', 4 ', 5' or 6 'positions of the sugar or modified sugar, including the 3' position of the sugar at the nucleotide at the 3 'end or the 5' position of the nucleotide from the 5 'end.

Modified sugars also include sugar mimetics, such as cyclobutyl residues or

IMPI

2 MEXICAN INSTITUTE

OF THE FROFIEDAD

INDUSTRIAL cyclopentyl, instead of pentofuranosyl sugar. Representative US patents describing the preparation of such modified sugar structures include, but are not limited to, US Patents.

No.: 4,981,957; 5,118,800; 5,319,080 and 5,359,044. Some of the modified sugars considered include:

^R\^^to

R³ R²^Rii ^^{I know}^^Ba *^ιγ-γ^Β3^RW

R³ R²

R³ R²^3nd R³ R²

Q = Me, Et, z'-Pr.

Other non-limiting examples of modified sugars include glycerol, which forms glycerol nucleic acid analogs (GNA). An example of a GNA analog is provided below, which is described in Zhang, R et al., J.

Am. Chem. Soc., 2008, 130, 5846-5847; Zhang L, et al., J. Am.

Chem. Soc., 2005, 127, 4174-4175 and Tsai CH et al. , PNAS,

2007, 14598-14603:

Τ 'o

o = p — oo

O = P-0 I

X

Bs where X is as defined herein. Another example of a GNA-derived analog, flexible nucleic acid (FNA) based on the mixture of amino acetal of formyl glycerol, is described in Joyce GF et al. , PNAS, 1987,

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INSTITUTO MSJdCANü '

DS THE PROPERTY

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84, 4398-4402 and Heuberger BD and Switzer C, J. Am. Chem. Soc.,

173

2008, 130, 412-413, which is provided below:

io

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or

Other non-limiting examples of modified sugars include hexopyranosyl sugars (6 'to 4'), pentopyranosyl 10 (4 'to 2'), pentopyranosyl (4 'to 3'), or tetrofuranosyl (3 'to

2' ) .

The hexopyranosyl sugars (6 'to 4') that are considered

<img file="MX342945B_D0358.tif" />

pentopyranosyl

Those considered sugars include:

(4 'to 2' that

<img file="MX342945B_D0359.tif" />

<img file="MX342945B_D0360.tif" />

JWV

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<img file="MX342945B_D0362.tif" />

174

The pentopyranosyl sugars (4 'consider

They consider them include:

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a 3 ') &lt;? W^TWAtI know

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ΛΛ / V sugars include:

tetrofuranosyl (3 'to 2' that

<img file="MX342945B_D0364.tif" />

Other modified sugars considered include:

<img file="MX342945B_D0365.tif" />

Additionally, the sugar mimetics illustrated below are considered, where X is selected from S,

Se, CH2, N-Me, N-Et or N-iPr.

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Sugars and modified sugar mimetics can be prepared by methods known in the art, including, but not limited to: A. Eschenmoser, Science (1999), 284: 2118; M. Bohringer (1992), 75: 1416-1477; M. Egli (2006), 128 (33): 10847-56, - ​​A.

Synthesis: Gnosis to Prognosis,

Sniekus, Ed., (Kluwer Academic,

K.-U. Schoning et al, Science et al, Helv. Chim. Acta et al, J. Am. Chem. Soc.

Eschenmoser in Chemical

C. Chatgilialoglu and V.

Netherlands, 1996), p.293;

(2000), 290: 1347-1351; TO.

<td></td><td></td><td></td><td> 176</td><td></td><td></td>

<td>Eschenmoser</td><td>et</td><td>to the,</td><td>Helv. Chim.</td><td>Minutes</td><td> (3.992) ,</td>

<td>Hunziker et</td><td>to the,</td><td>Helv.</td><td>Chim. Minutes</td><td> (1993) ,</td><td> 76:259;</td>

<td colspan="2">Al, Helv. Chim.</td><td>Minutes</td><td colspan="2">(1993), 76: 2701; K.</td><td>Groebke</td>

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

Chim. Acta (1998), 81: 375 and A. Eschenmoser, Science (1999),

284:2118.

Blocking groups

In the described reactions, in certain modalities it is necessary to protect reactive functional groups, for example, hydroxy, amino, thiol or carboxy groups, when these are desired in the final product, to avoid their undesired participation in the reactions. The protecting groups are used to block some or all of the reactive residues and to prevent the participation of these groups in chemical reactions until the protecting group is removed. In one embodiment, each protecting group is removed through a different medium. Protective groups that are cleaved under completely disparate reaction conditions meet the differential suppression requirement. In some embodiments, the protecting groups are removed by an acid, a base, and / or hydrogenolysis. Groups such as trityl, dimethoxytryl, acetal, and t-butyldimethylsilyl are acid labile and are used in certain embodiments to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with groups.

177

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MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

<img file="MX342945B_D0368.tif" />

Cbz, which are removed by hydrogenolysis, and / or Fmoc groups, which are base labile. In other embodiments, the reactive carboxylic acid and hydroxy acid residues are blocked with acid-labile groups, such as, but not limited to, methyl, ethyl, and acetyl in the presence of amines blocked with acid-labile groups, such as, t-butylcarbamate or with carbamates that are both acid and base stable but can be hydrolytically removed.

In another embodiment, reactive hydroxy residues are blocked with protecting groups that are hydrolytically removed, such as the benzyl group, while amine groups that can bind hydrogen by acids are blocked with base-labile groups, such as Fmoc . In another embodiment, the reactive carboxylic acid residues are protected by conversion to simple ester compounds or, in another embodiment, blocked with protective groups that are oxidatively removed, such as 2,4-dimethoxybenzyl, while amino groups Coexisting are blocked with fluoride-labile silyl or carbamate blocking groups.

Allyl blocking groups are useful in the presence of acidic and basic protecting groups since the former are stable and can be subsequently removed by metal catalysts or pi acids. For example, an allyl-blocked hydroxy group can be deprotected with a

178

<img file="MX342945B_D0369.tif" />

Pd (0) catalyzed reaction in the presence of acid labile t-butylcarbamate protecting groups or base labile amine acetate. Another form of protecting group is a resin to which a compound or intermediate is added. While the residue is added to the resin, this functional group is blocked and cannot react. Once released from the resin, the functional group can react.

The most common blocking / protecting groups useful in the synthesis of the compounds described herein are, by way of example only:

or

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allyl ^v or

Cbi ajkn Me

B

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Bo &lt;

(H₃DC'

I-butyl

H, C CH,

V (H₃ C), C 'φτTBBMS

HjCÜ oMBn taritilo

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(CíHsJjC- ^

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Representative protecting groups useful for protecting nucleotides during synthesis include base-labile protecting groups and acid-labile protecting groups. Base labile protecting groups are used to protect exocyclic amino groups from heterocyclic nucleobases. This type of protection,

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179 &lt;^ mm) M_EXICAWOOF THE PR CINEDa or 'N £&gt; UST »f * i generally, is achieved by acylation. Three acylation groups commonly used for this purpose benzoyl chloride, phenoxyacetic anhydride, and isobutyryl chloride. These protecting groups are stable under the reaction conditions used during nucleic acid synthesis and cleave at approximately equivalent rates during base treatment at the end of synthesis.

In some embodiments, the 5 'protecting group is trityl, monomethoxy trityl, dimethoxytryl, trimethoxytryl, 2-chlorotryl, DATE, TBTr, 9-phenylxanthine-9-yl (Pixyl), or 9 (p-methoxyphenyl) xanthine-9-yl (MOX ).

In some embodiments, thiol residues are incorporated into compounds of Formula 1, 2, 4, or 5 and protected. In some embodiments, protecting groups include, but are not limited to, pixyl, trityl, benzyl, pmethoxybenzyl (PMB), or tere-butyl (t-Bu).

Other protecting groups, plus a detailed description of the techniques applicable to creating protective groups and removing them are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3^to Ed., John Wiley &amp;

Sons, New York, NY, 1999 and Kocienski, Protective Groups,

Thieme Verlag, New York, NY, 1994, which is incorporated herein by reference for its disclosure.

180

<img file="MX342945B_D0375.tif" />

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MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL

METHODS OF USE OF NUCLEIC ACID DRUGS

WHICH INCLUDE A REST OF CHIRAL X-PHOSPHONATE

The stereodefined oligonucleotide prodrug comprising a chiral phosphorous or phosphorothioate moiety obtained by the methods of the invention is useful in a variety of areas for its application, due to a combination of stability, defined chirality, and ease of synthesis. Generally speaking, compounds synthesized by this method are useful as therapeutic products, diagnostic probes or reagents, synthetic tools to produce other oligonucleotide products, and nanostructure materials suitable for a variety of new material and computer applications.

The stereodefined oligonucleotide prodrug of the invention possesses better stability in serum compared to that of natural DNA / RNA equivalents and, in particular, the stereodefined oligonucleotide prodrug belonging to the phosphorothioate class. Furthermore, the SP isomer is more stable than the RP isomer. In some modalities, the serum stability level is modulated by introducing all SP centers or SP centers at selected positions to provide resistance to degradation. In other modalities, the introduction of the RP and / or SP stereo centers that can be

181

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

Γ &lt;^Α'· ^' Ϊ́'ιπ⁵ selecting can provide a specific base pairing association with an endogenous or exogenous target, thereby protecting the target from metabolism or improving a particular biological reaction.

RNase H activation is also modulated by the presence of stereocontrolled phosphorothioate nucleic acid analogs, where natural DNA / RNA is more susceptible than the RP stereoisomer which, in turn, is more susceptible than the corresponding SP isomer.

Increased stability of the duplex to RNA is observed with the phosphorothioate RP oligonucleotides that have greater duplex stability than the corresponding SP oligonucleotides which, in turn, demonstrate greater stability than that of native DNA / RNA.

An increase in the stability of the duplex towards the

DNA with SP that has higher duplex stability than RP that has more stability than natural DNA / RNA. (P. Guga, Curr. Top Med. Chem., 2007, 7, 695-713).

These molecules can be useful as therapeutic agents in a variety of particular applications. They can be incorporated into oligonucleotides that also contain the standard DNA / RNA nucleosides or can be synthesized as complete sequences of the stereocontrolled oligonucleotides of the invention. Some categories of

182

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MEXICAN INSTITUTE OF THE INDUSTRIAL PKOMÉÜAD

<img file="MX342945B_D0376.tif" />

Therapeutic agents include, but are not limited to, antisense oligonucleotides, antigen oligonucleotides that form a triple helix with target sequences to suppress transcription of unwanted genes and modulate protein expression and / or activity, decoy oligonucleotides, DNA vaccines , aptamers, ribozymes, deoxyribozymes (DNA enzymes or DNA enzymes), siRNA, micro

P-modified RNA, cRNA (non-coding RNA), and prodrugs. Modulation directly or indirectly involves increasing or decreasing the activity of a protein or inhibiting or promoting the expression of a protein. These nucleic acid compounds can be used to control cell proliferation, viral replication, or any other cell signaling process.

In one example, the scope of siRNA therapeutic products requires oligonucleotide species that allow greater stability against RNase activity, in order to improve the duration of action over that seen with the siRNA composed of natural nucleosides. Additionally, the formation of A-helices appears to indicate success in entering RNAi rather than the presence of specific natural elements in the oligonucleotide. Both of these requirements, which can be provided through the use of stereocontrolled oligonucleotides of

183

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OF THE PROPERTY &lt;*&gt; »« 2S8Í. INDUSTRIAL invention, can provide greater stability (YL Chiu,

TM Rana RNA, 2003, 9,1034-1048).

Treatment methods

The nucleic acids described herein are useful as therapeutic agents against various disease states, including their use as antiviral agents. Nucleic acids can be used as agents for the treatment of diseases through modulation of DNA and / or RNA activity. In some embodiments, nucleic acids can be used to inhibit specific gene expression. For example, nucleic acids can be complementary to a specific target messenger RNA (mRNA) sequence. They can be used to inhibit the viral replication of thousands of viruses. Examples of virus families include orthomyxovirus, poxvirus, herpesvirus, papillomavirus, picornavirus, flavivirus, retrovirus, hepatitis virus, paramyxovirus, reovirus, parvovirus, phylovirus, coronavirus, arenavirus, rhabdovirus, and adenovirus. Additional virus families are known and considered herein. Other examples include uses as antisense compounds against HIV RNA or other retroviral RNA or to hybridize to HIV mRNA encoding the tat protein or to the TAR region of HIV mRNA. In some embodiments, nucleic acids mimic the secondary structure of the TAR region of the ιμριγ ^ mRNA ^

4

MEXICAN INSTITUTE 1

D £ INDUSTRIAL PROPERTY

<img file="MX342945B_D0378.tif" />

HIV and therefore bind to the tat protein. In one embodiment, nucleic acids are used to inhibit the expression of a target protein by contacting a cell with a compound of Formula 1, where the expression of other proteins in the cell is either not minimally inhibited or inhibited. In one embodiment, inhibition of the target protein occurs in vivo in a mammal. In other embodiments, a therapeutically effective amount of a compound of Formula 1 is administered to inhibit expression of a target protein.

Other examples of proteins where expression can be modulated include Jun N-terminal kinase (JNK) proteins, diacylglycerol acyltransferase I, apolipoprotein B, glucagon receptor, Aurora B, acyl CoA cholesterol acyltransferase-2, c-reactive protein, STAT (signal transducers and transcription activators) family of proteins and MDR glycoprotein P. Nucleic acids can be used to inhibit the expression of protein phosphatase IB (PTP1B), RNA polymerase dependent on viral RNA. Nucleic acids can be used to induce events such as apoptosis in cancer cells or to make a cell more susceptible to apoptosis. Nucleic acids can be used to modulate the activities of proteins. For example, they can help

185

ΙΜΡφ

INSTITUTO MEXICAN 1 DE LA PROPIEDAD INDUSTRIAL modulate RNase H activity directed at multi-drug resistance (MDR) RNA molecules.

In another aspect, the present invention provides methods of treating a disease mediated by unwanted gene expression in a subject (eg, mammals, such as humans) in need of such treatment. By diseases is meant diseases or symptoms of diseases. The method includes administering to the subject an effective amount of a non-racemic prooligonucleotide of the present invention.

Examples of diseases mediated by unwanted gene expression include cancer (eg, leukemia, tumors, and metastasis), allergy, asthma, obesity, inflammation (eg, inflammatory diseases, such as, inflammatory airway disease) , hypercholesterolemia, hematological disorders, severe acute respiratory syndrome (SARS), obstructive airways disease, asthma, autoimmune diseases, retroviral diseases, such as, AIDS or HIV, other viral infections, intrauterine infections, metabolic diseases, infection (eg, bacterial, viral, yeast, fungal), CNS diseases, brain tumors, degenerative neuronal diseases, cardiovascular diseases, and diseases associated with angiogenesis, neovascularization, and vasculogenesis.

186 τ χ &lt;η τ

<img file="MX342945B_D0379.tif" />

In an exemplary embodiment, the compounds are useful in treating cancer, including pancreatic cancer and other diseases or disorders involving abnormal cell proliferation.

Located in the upper abdomen (in the retroperitoneum), the pancreas is a dual-function gland of the digestive and endocrine systems. In some cases, the pancreas works like an endocrine gland (eg, producing several important hormones). In some cases, the pancreas works like an exocrine gland (eg, secreting fluids that contain digestive enzymes that pass into the small intestine).

Pancreatic cancer is the fourth most common cause of cancer death in the United States (after lung, colon, and breast cancer), accounting for 6% of all cancer deaths. In 2008, 37,680 new cases of pancreatic cancer will have been diagnosed in

United States, with 34,290 deaths. The disease rate increases linearly after age 50, where the only definitive risk factor is cigarette smoking (smokers are four times more likely to develop the disease than non-smokers). Invasive pancreatic cancer is practically always fatal. Collective mean survival time

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EU MEXICAN INSTITUTE INDUSTRIAL PROPERTY

187 fesí¿ of all patients is 4 to 6 months. The 1-year relative survival is 24%, the overall 5-year survival rate is &lt;5%.

Pancreatic cancer is asymptomatic in its first stage and is generally undiagnosed for several months (less than a third of patients are diagnosed before the end of the 2 months after the onset of initial symptoms). In some cases, delayed diagnosis results (partially or completely) in metastasis of cancer cells in the liver or lymph nodes.

Currently, surgery (resection of the pancreas) is the main therapy and the only cure for pancreatic cancer. However, only 15 to 25% of tumors can be removed at the time of diagnosis, and only 10 to 20% of patients who undergo surgery survive more than two years. Once infiltration of the tumor has occurred and other tissues have been affected, surgery is no longer possible.

In some cases, diabetes mellitus or pancreatitis predisposes the individual to develop a proliferative disorder of a plurality of pancreatic cells. In certain cases, individuals are at increased risk of developing a proliferative disorder of a

188

ΙΜΡΙ

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<img file="MX342945B_D0380.tif" />

a plurality of pancreatic cells due to an inherited syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer (CCHNP) and familial adenomatous polyposis (PAF). In some cases, individuals are at increased risk of developing a proliferative disorder of a plurality of pancreatic cells due to the mutation in a gene that is selected from the group consisting of MSH2, MSH6, MLH1, and APC.

Ideally, effective treatment for pancreatic cancer (i) should control the main tumor mass, both initially and subsequently, and (ii) treat metastatic tumor cells. Chemoprophylaxis (the administration of agents such as drugs, biologics, nutrients, and the like) slows the progression, reverses, or inhibits carcinogenesis, thereby decreasing the risk of developing an invasive or clinically significant disease.

In certain embodiments herein, a method of treating pancreatic cancer is described. As used herein, pancreatic cancer includes forms of cancer of the pancreas. In some modalities, pancreatic cancer is metastatic pancreatic cancer. In some modalities, pancreatic cancer is a carcinoma, sarcoma, cancer, or combinations of these. In some modalities, cancer

189

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MEXICAN INSTITUTE Dt THE INDUSTRIAL PROPERTY

<img file="MX342945B_D0381.tif" />

Pancreatic cancer treated includes sporadic and inherited pancreatic cancers. In some modalities, pancreatic cancer is duct cell carcinoma, acinar cell carcinoma, papillary mucinous carcinoma, signet ring carcinoma, adeno-squamous carcinoma, undifferentiated carcinoma, mucinous carcinoma, giant cell carcinoma, small cell carcinoma, cystic cancer , serous cystic cancer, mucinous cystic cancer, unclassified pancreatic cancer, pancreatoblastoma, or combinations thereof.

In some modalities, an individual in need of pancreatic cancer treatment has a localized tumor of the pancreas. In some modalities, an individual in need of pancreatic cancer treatment presents a biopsy without involvement of the regional lymph nodes. In some modalities, an individual who needs pancreatic cancer treatment presents a biopsy with involvement of the regional lymph nodes. In some modalities, an individual needing pancreatic cancer treatment presents with a negative nodular pancreatic tumor (eg, no nodular involvement). In some modalities, an individual in need of pancreatic cancer treatment presents with a positive nodular tumor (eg, with nodular involvement).

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190 MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL ^ ¿7 * ^ lz&gt;

In some modalities, pancreatic cancer in an individual in need of pancreatic cancer treatment metastasizes elsewhere in the body. In some modalities, pancreatic cancer metastasizes to a site selected from the group consisting of lymph nodes, stomach, bile duct, liver, bone, ovary, peritoneum, and brain.

In some embodiments, cancer cells or precancerous cells are identified through typing or histological evaluation of a tissue sample (eg, a biopsy sample). In some embodiments, cancer cells or precancerous cells are identified through the use of suitable molecular markers.

In some modalities, pancreatic cancer in an individual needing pancreatic cancer treatment is staged according to the classification system of the

American Joint Commission on Cancer (AJCC) for classification of malignant tumors (TNM), where the tumor (T) has been assigned a stage of Tx, TI, T2, T3, T4; and where the regional lymph nodes (N) have been assigned a stage of NX, NO, NI; and where distant metastases (M) have been assigned a stage of MX, M0 or MI. In some modalities, pancreatic cancer in an individual who

IMPI needs pancreatic cancer treatment is staged as

191 Stage 0, I, IA, IB, II, HA, IIB, III and IV pancreatic cancer. In some modalities, pancreatic cancer in an individual needing pancreatic cancer treatment is staged as Grade GX (eg, grade cannot be assessed), Grade 1, Grade 2, Grade 3, or Grade 4.

More specific examples of cancers that are treated with the compounds of the present invention include breast cancer, lung cancer, melanoma, colorectal cancer, bladder cancer, ovarian cancer, prostate cancer, kidney cancer, squamous cell cancer, glioblastoma, Kaposi's sarcoma, multiple myeloma, and leukemia.

Cancer evaluation and treatment

The term tumor cell antigen is defined herein as an antigen that is present in greater quantities in a tumor cell or in body fluids than in unrelated tumor cells, normal cells, or normal body fluids. The presence of antigens can be tested by any number of assays known to those of skill in the art and include, but are not limited to, negative and / or positive antibody selection, such as an ELISA assay, a radioimmunoassay, or by blotting Western.

192

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<img file="MX342945B_D0382.tif" />

The apoptosis-inducing agent is defined herein as an agent that induces apoptosis / programmed cell death and includes, for example, anticancer agents and treatments where cells (eg, tumor cells) are induced to undergo cell death scheduled. Examples of apoptosis-inducing agents are described in more detail below.

The terms apoptosis or programmed cell death refer to the physiological process by which unwanted or unusable cells are killed during development and other normal biological processes. Apoptosis is a form of cell death that occurs under normal physiological conditions, where the cell actively participates in its own disappearance (cell suicide). It is most often found during normal cell regeneration and tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance, development of the nervous system, and atrophy of dependent endocrine tissue. Cells undergoing apoptosis exhibit characteristic morphological and biochemical features. These features include chromatin aggregation, nuclear and cytoplasmic condensation, division of the cytoplasm and nucleus into membrane-bound vesicles (apoptotic bodies), which contain ribosomes, morphologically intact mitochondria, and

<img file="MX342945B_D0383.tif" />

193 ΙΜΡΙ

MEXICAN INSTITUTE

Say THE PROPERTY nuclear material. In vivo, these apopt bodies are rapidly recognized and phagocytosed by macrophages, dendritic arteries, or adjacent epithelial cells. Due to this effective mechanism for the elimination of apoptotic cells in vivo, an inflammatory response is not elicited. In vitro, apoptotic bodies, as well as the remaining cell fragments, ultimately swell and eventually lysate. This terminal phase of in vitro cell death is called secondary necrosis. Apoptosis can be measured by methods known to those of skill in the art such as DNA fragmentation, Annexin V exposure, caspase activation, cytochrome c release, etc. A cell that has been induced to disappear is referred to herein as an apoptotic cell.

Apoptosis can also be tested using a

Standard Annexin V Apoptosis Assay: NIH: OVCAR-3 cells were grown in 6-well plates (NUNC) and irradiated or treated with an antagonist (or in combination with another anticancer drug) for 4 to 48 hours, washed and stained with Annexin V-FITC (BD-Pharmingen) for 1 hour.

Cells are analyzed by flow cytometry (Becton-Dickinson, CellQuest), counterstained with propidium iodide and analyzed again on the flow cytometer.

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INSTITUTO MBXICANO fT * • E LA TROflEWAD

INDUSTRIAL&gt; * l

Patients can be analyzed for symptoms at one or more multiple times, including before, during, and after treatment regimens. Treatment can result in improvement of the subject's condition and can be analyzed by determining whether one or more of the following factors have occurred:

decreased tumor size, decreased cell proliferation, decreased cell numbers, decreased neovascularization, increased apoptosis, or decreased survival of at least a portion of tumor cells. One or more of these occurrences, in some cases, can result in partial or total elimination of the cancer and prolongation of the patient's survival. Alternatively, for end-stage cancers, treatment can result in disease stasis, a better quality of life, and / or prolonged survival.

Cell migration assay methods

Assays for cell migration have been described in the literature, eg. , Brooks, et al. , J. Clin. Invest

1997, 99: 1390-1398 and methods for measuring cell migration are known to those of skill in the art. In a method for measuring cell migration described herein, the membranes of transwell migration chambers are

195

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0384.tif" />

cover with substrate, transwells are washed, and non-specific binding sites are blocked with BSA. Tumor cells from the sub-confluent cultures are collected, washed and resuspended in migration buffer in the presence or absence of test antibodies.

After the tumor cells are allowed to migrate to the bottom of the covered transwell membranes, the cells that remain at the top of the membrane are removed and the cells that migrate to the bottom are stained with gentian violet. Then, cell migration is quantified by direct cell counts by microscopic field.

Tumor growth test methods

Tumor growth can be assayed by methods known to those skilled in the art, eg, the SCID mouse model, the hairless mouse model, and BALB / c mice with syngeneic tumors. SCID mouse models for tumor growth are performed as follows: Subconfluent human M21 melanoma cells (or any desired tumor cell type) are harvested, washed, and resuspended in sterile PBS (20 x 106 per mL). SCID mice are injected subcutaneously with 100 pL of a suspension of M21 human melanoma cells (10 6 times). Three days after

196

IMPI

INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL injection of tumor cells, mice are not treated or are treated intraperitoneally with an antagonist at the desired dose intervals. Mice are treated daily for 24 days. Tumor size is measured with calipers and the volume estimated using the formula V = (L x W2) / 2, where V is equivalent to volume, L is equivalent to length, and W is equivalent to width.

Alternatively, hairless mouse models, SCID mouse models, and / or BALB / c syngeneic mouse models can also be used to assess tumor growth and inhibition thereof by humanized anti-endoglin antibodies or antigen-binding fragments described herein.

Cell proliferation assay methods

Cell proliferation can be assayed by methods known to those skilled in the art. As described herein, subconfluent human endothelial cells (HUVEC) can be resuspended in a proliferation buffer containing low serum level (5%) in the presence or absence of CM (25 pL) of ECV cells o ECVL and endothelial cells are allowed to proliferate for 24 hours. Proliferation can be quantified by measuring mitochondrial dehydrogenase activity using a WST-1 assay kit (Chemicon) available from the

<img file="MX342945B_D0385.tif" />

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INDUSTRIAL

<img file="MX342945B_D0386.tif" />

Commerce. Furthermore, as described herein, proliferation can be quantified by measuring 3H incorporation using standard methods. (She et al., Int. J.

Cancer, 108: 251-257 (2004)).

Other methods for evaluating cell proliferation are known in the art and are considered herein. Additional non-restrictive examples are described in more detail in the examples.

It is understood that the classification and staging systems described herein represent a means of evaluating the treatment of cancers described herein; additionally, other staging schemes are known in the art and can be used in connection with the methods described herein. By way of example only, the TNM classification of malignant tumors can be used as a cancer staging system to describe the extent of cancer in a patient's body. T describes the size of the tumor and if it has invaded nearby tissue, N describes the regional lymph nodes involved, and M describes distant metastases. The International Union Against Cancer (UICC) preserves the TNM and is used by the Joint Commission

American Cancer Association (AJCC) and the Federation

International Gynecology and Obstetrics (FIGO). It is understood that not all tumors have TNM classifications,

198

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<img file="MX342945B_D0387.tif" />

such as, for example, brain tumors. In general, T (a, is, (0), 1-4) is measured as the size or direct range of the primary tumor. N (0-3) refers to the degree of spread to regional lymph nodes: it does NOT mean that tumor cells are absent from regional lymph nodes, nor does it mean that tumor cells spread to the closest or least numerous regional lymph nodes. , N2 means that tumor cells spread in an NI to N3 range; N3 means that tumor cells spread to the most distant or numerous regional lymph nodes. M (0/1) refers to the. presence of metastases: M0 means that no distant metastases are present; ML means that metastases occurred in distant organs (beyond regional lymph nodes). Other parameters can also be evaluated. G (1-4) refers to the grade of cancer cells (that is, they are low grade if they appear similar to normal cells and high grade if they appear poorly differentiated). R (0/1/2) refers to the entirety of an operation (that is, the resection limits are free of cancer cells or not). L (0/1) refers to the invasion of the lymphatic vessels. L (0/1) refers to the invasion of the veins. C (1-4) ago

199

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INSTITUTO MEXICANO OE LA PROPIEDAD reference to a certainty modifier (quality and^NOTí5gr "V

Methods to degrade are provided herein,¹ inhibiting the growth of or killing cancer cells, which comprise contacting the cells with an amount of a compound described herein effective to degrade, inhibit the growth of or kill cancer cells.

Methods are provided herein for inhibiting tumor size growth, reducing tumor size, reducing tumor proliferation, or preventing tumor proliferation in an individual, which comprises administering to said individual an effective amount of a compound described in herein to inhibit growth of tumor size, reduce tumor size, reduce tumor proliferation, or prevent tumor proliferation. In some cases, the treatment of tumors includes stasis of symptoms, that is, by treating the patient, the cancer does not worsen and the patient's survival is prolonged.

Patients can be evaluated for symptoms at one or more multiple times, including before, during, and after treatment regimens. Treatment can result in improvement of the subject's condition and can be assessed by determining whether

<img file="MX342945B_D0388.tif" />

200

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MEXICAN INSTITUTE

<img file="MX342945B_D0389.tif" />

DELA MONEDAD INDUSTRIAL produced one or more of the following events: decrease in tumor size, decrease in the proliferation of tumor cells, decrease in the number of cells, decrease in neovascularization and / or increase in apoptosis. One or more of these occurrences, in some cases, can result in partial or total elimination of the cancer and prolongation of the patient's survival. Alternatively, for end-stage cancers, treatment may result in disease stasis, better quality of life, and / or prolonged survival. Other methods of evaluating treatment are known in the art and are considered herein.

In an exemplary embodiment, the pro15 oligonucleotide compounds of the invention are administered to a subject, such as a mammal (eg, a human) suffering from a medical disorder, eg, cancer or non-malignant conditions characterized by the presence of a class of unwanted cells.

Initial outcome measures can be evaluated for patients who are treated using the methods described herein, including, for example, progression-free survival. In one modality, an evolution-free increase in survival is observed in

201

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INDUSTRIAL an amount of about 2 times, 5 times, 10 times

<td>20 times, 50</td><td>times</td><td>or more</td><td>, compared</td><td>with the</td><td>lack</td><td>of</td>

<td>treatment.</td><td colspan="2">In other</td><td>modality, a</td><td>increase</td><td>of</td><td>the</td>

<td>survival</td><td>free</td><td>of</td><td>evolution is the</td><td>increase</td><td>of</td><td>the</td>

Survival in approximately 3 months, approximately 6 months, approximately 9 months, approximately 12 months, approximately approximately months, approximately 2 years, years, approximately 4 years, approximately 5 years or more compared to the lack of treatment.

Secondary outcome measures can also be assessed, including duration of response, tumor progression relative to time, overall survival, serious and non-serious adverse events. For example, a treatment can prevent disease progression (i.e., stasis) or may result in improvement. Alternatively, or in addition, other targets may be measured with respect to one or more of the following: decreased tumor burden, decreased neovascularization, reduced side effects, decreased adverse reactions, and / or increased patient compliance.

Other specific examples of diseases or disorders for which treatment by

IMPI ^

MIXICAN INSTITUTE * χ · * “ϊϊ &amp; 3

OF THE FÜDPÍEDAD

0 2 INDUSTRIAL compounds or compositions of the invention-paia SO treatment or prevention include, but are not limited to, transplant rejection (eg, kidney, liver, heart, lung, islet cells, pancreas, bone marrow, cornea, small intestine, allografts or xenografts of the skin and other transplants), graft versus host disease, osteoarthritis, rheumatoid arthritis, multiple sclerosis, diabetes, diabetic retinopathy, inflammatory bowel disease (eg. , Crohn's disease, ulcerative colitis and other intestinal diseases), kidney disease, cachexia, septic shock, lupus, myasthenia gravis, psoriasis, dermatitis, eczema, seborrhea,

Alzheimer's, Parkinson's disease, protection of stem cells during chemotherapy, ex vivo selection or ex vivo clearance for autologous or allogeneic bone marrow transplantation, eye disease, retinopathies (eg, macular degeneration, diabetic retinopathy and other retinopathies) , corneal disease, glaucoma, infections (eg, bacterial, viral or fungal), heart disease, among others, restenosis.

RNAse L activation

The 2'-5 'oligoadenylate (2-5A) / RNase L pathway is one of the enzyme pathways induced by interferon. Rnasa L is activated after binding to the 5 'phosphorylated fragments

IMPI ^

- _ MEXICAN INSTITUTE +

03 ΓΗ THE PROPERTY í

INDUSTRIAL 2'-5 'Adenylic Acid. These fragments of 2'-5 'adenyl acid (2-5A) are produced under the control of 2'-5' oligo (A) synthetase. This pathway is part of the innate immune system and plays an important role in preventing a viral infection. 2-5A-induced cleavage of single-stranded RNA results in apoptosis. Biostable phosphorothioate analogs of 2-5A have been shown to be potent activators of Rnasa L (Xianh et al.,

Cancer Research (2003), 63: 6795-6801). In the present study, 2-5A analogues induce Rnasa L activity and cause apoptosis in cultures of advanced stage metastatic human prostate cancer cell lines.

DU145, PC3 and LNCaP.

Sustained activation of RNase L releases a mitochondrial apoptosis pathway that removes virus-infected cells as well as cancer / tumor cells. The

RNase L can inhibit fibrosarcoma growth, prostate cancer growth, colorectal cancer growth, and pancreatic cancer growth. Due to the usual role of RNase L in different cancers, it is considered that the invention described herein can be used for the treatment of any type of cancer.

Silverman, RH, Cytokine Growth Factor Rev, 18 (5-6): 381-388 (2007); Bisbal, C. and Silverman, RH, Biochimie. 89 (6-7): 789-

<img file="MX342945B_D0390.tif" />

204

INDUSTRIAL

798 (2007). By way of example, downregulation of RNase L refers to any reduction in the expression levels of the gene or genes encoding RNase.

L, silencing of the gene or genes encoding RNase L, reduction of expression / translation levels of proteins comprising RNase L, reduction of the amount of RNase L present within a cell and / or any reduction in activity of RNase L compared to a predetermined level of RNase L in a healthy population taken as an example. Alternatively, any reduction in RNase L levels, as described herein, may indicate downregulation of RNase L.

In an exemplary embodiment, the compounds described herein are useful for the treatment of diseases possessing down-regulated RNase L. In another embodiment, the disease associated with down-regulated RNase L is cancer. In additional modalities, cancer

<td>is</td><td>Cancer</td><td>pancreatic cancer</td><td>of</td><td>prostate or</td><td>Cancer</td>

<td colspan="2">20 colorectal.</td><td>Alternatively,</td><td>the</td><td colspan="2">described compounds</td>

<td>in</td><td colspan="2">these are useful for</td><td>the</td><td>treatment</td><td>of a</td>

disease that has down-regulated RNase L. In one embodiment example, the disease possessing down-regulated RNase L is chronic fatigue syndrome.

ΙΜΡΙ

205 'NSTITUTO MEXICa Ni i Dt THE PROPERTY

INDUSTRIAL ΧζΓ'Τβ ^ Μέ.

Additional diseases possessing regulated RNase L-pwaumento are known in the art and are considered herein.

When used as a therapeutic product, the nucleic acid described herein is administered as a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a nucleic acid comprising a chiral X-phosphonate moiety of Formula 1 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable inactive ingredient selected from pharmaceutically acceptable diluents, pharmaceutically acceptable excipients and pharmaceutically acceptable carriers. In another embodiment, the pharmaceutical composition is formulated for intravenous injection, oral administration, buccal administration, inhalation, nasal administration, topical administration, ophthalmic administration, or otic administration. In further embodiments, the pharmaceutical composition is a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, ointment, lotion, eye drops, or ear drops.

206

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<img file="MX342945B_D0391.tif" />

Pharmaceutical Compositions and Administration

In another aspect, the present invention provides a pharmaceutical composition comprising a non-racemic pro-oligonucleotide mixed with a pharmaceutically acceptable carrier. One skilled in the art will recognize that pharmaceutical compositions include the pharmaceutically acceptable salts of the non-racemic pro-oligonucleotides described above.

Compounds to increase and direct administration

The pro-oligonucleotides described herein can be administered using a variety of administration strategies, including, but not limited to, oligonucleotide conjugates with various ligands, as well as the use of nanocarriers. All nucleic acid delivery strategies are contemplated for use with the prooligonucleotides described herein. The choice between examples of administration strategies, which include, but are not limited to, chemical conjugates, lipid / liposomal cation transfer vesicles, and supramolecular nanocarriers, depend on the therapeutic context and the methods for determining the ideal administration modality are known in the technique and are considered herein.

Cell penetrating compounds (CPC)

Various compounds are known to act as carriers

207

<img file="MX342945B_D0392.tif" />

<img file="MX342945B_D0393.tif" />

fillers, such as nucleic acids, and facilitate entry of nucleic acid into a cell in an in vivo configuration. Examples of carriers are described in Dietz et al. , Molecular &amp; Cellular Neuroscience, 27 (2): 85-131, which is incorporated herein by this reference. The

Prototypic CPCs derived from Tat and from antennepedia transcriptional regulators have been linked by a large number of new residues. As an example, CPCs that are peptides can be relatively short polycationic peptides (9 to 30 amino acids), rich in arginine and lysine, or hydrophobic sequences that interact with the membrane. The

CPCs can be linked by recombinant DNA techniques or can be chemically coupled to peptides, oligonucleotides, or nanocarriers, thus comprising the charge of CPCs.

Cell-Directed Ligands (CTL)

Another strategy is to administer oligonucleotides using CTLs that bind with high affinity to a cell surface receptor that can undergo efficient internalization. Potential ligands include antibodies, polypeptides derived from phage display libraries, and small organic molecules. Other cell-directed ligands are known in the art or will be developed, which are considered for use with the invention described herein. Because to

208

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<img file="MX342945B_D0394.tif" />

Often several receptors are preferentially expressed in particular cell types, this approach offers the possibility of improving the selectivity of oligonucleotide reagents. Examples of target receptors include, but are not limited to, lipoprotein receptors (such as those found in the liver), integrins, receptor tyrosine kinases, and the G protein-coupled receptor (GPCR) superfamily.

Nanocarriers

A variety of supramolecular nanocarriers can be used to deliver nucleic acids. Examples of nanocarriers include, but are not limited to, liposomes, cationic polymer complexes, and various polymers. Nucleic acid complex formation with various polycations is another approach to intracellular administration, this includes the use of PEGylated polycations, polyethylene amine (PEI) complexes, cationic block copolymers and dendrimers. Various cationic nanocarriers, including PEIs and polyamidoamine dendrimers help to release the contents of the endosomes. Other approaches include the use of polymeric nanoparticles, polymer micelles, quantum dots, and lipoplexes.

In addition to the examples of administration strategies described herein, other strategies are known.

209 I jMí Ρ I

INSTITUTO MEXICANO DE LA NIONEDAD for the administration of nucleic acids. Nduxtrial

In therapeutic and / or diagnostic applications, ios

<img file="MX342945B_D0395.tif" />

Compounds of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. In general, the techniques and formulations can be found in Remington,

The Science and Practice of Pharmacy, (20^to ed. 2000).

The compounds according to the invention are effective over a wide dosage range. For example, in the treatment of adult humans, dosages of 0.01 to

1000 mg, 0.5 to 100 mg, 1 to 50 mg per day and 5 to

100 mg per day are examples of dosages that can be used. The exact dosage will depend on the route of administration, the way in which the compound is administered, the subject to be treated, the body weight of the subject to be treated and the preference and experience of the treating physician.

Pharmaceutically acceptable salts, in general, are known to those skilled in the art and include, by way of example but not limited to, acetate, benzenesulfonate, besilate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, carbonate, carbonate, citrate , edetate, edisylate, stolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolilarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate,

210

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<img file="MX342945B_D0396.tif" />

iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsilate, nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate . Other pharmaceutically acceptable salts can be found in, for example, Remington, The Science and Practice of Pharmacy (20^to ed. 2000). Preferred pharmaceutically acceptable salts include, for example, acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsilate, pamoate (embonate), phosphate, salicylate, succinate, sulfate or tartrate.

Depending on the specific conditions being treated, such agents can be formulated in liquid or solid dosage forms and administered systemically or locally. The agents can be administered, for example, in a low-time or sustained release mode, as is known to those skilled in the art. Formulation and administration techniques can be found in Remington, The Science and Practice of Pharmacy (20^to ed. 2000). Suitable routes may include oral, buccal, inhalation, sublingual, rectal, transdermal, vaginal, transmucosal, nasal, or intestinal administration; parental administration, among others,

INSTITUTO MEXICANC nt la propriedat intramuscular, subcutaneous, intramedullary injection, a^lS^Ti^{,TO THE}co'

211

<img file="MX342945B_D0397.tif" />

Intrathecal, direct intraventricular, intravenous, intraarticular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injection or other modes of administration.

For injection, the agents of the invention can be formulated and diluted in aqueous solutions, such as, physiologically compatible buffer solutions such as Hank's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, appropriate penetrants are used in the formulation to penetrate the barrier. In general, such penetrators are known in the art.

The use of pharmaceutically acceptable inert carriers to formulate the compounds described herein to practice the invention in dosages suitable for systemic administration is within the scope of the invention. With the proper choice of a carrier and the proper manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, can be administered parenterally, such as by intravenous injection. The compounds can be easily formulated using pharmaceutically acceptable carriers known in the

212

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INSTITUTO MEXICAN,) ÍJE LA MOHEDAL) INDUSTRIAL the technique in dosages suitable for oral administration. Such carriers allow the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject (eg, patient) to be treated.

For nasal or inhalation administration, the agents of the invention may also be formulated by methods known to those skilled in the art and include, but are not limited to, examples of solubilization, dilution, or dispersion of substances, such as, saline, preservatives, such as benzyl alcohol, absorption promoters and fluorocarbons.

Pharmaceutical compositions suitable for use in the present invention include compositions where the active ingredients are contained in an effective amount.

<td>for</td><td>achieve your</td><td colspan="2">desired goal.</td><td>The</td><td>experts in</td><td>The technique</td>

<td>are</td><td>able</td><td>of</td><td>decide</td><td>the</td><td>quantities</td><td>effective,</td>

<td colspan="2">especially</td><td>in</td><td>view of</td><td>the</td><td>description</td><td>detailed</td>

<td colspan="3">provided in the</td><td>Present.</td><td></td><td></td><td></td>

<td></td><td>In addition to</td><td>the</td><td>ingredients</td><td colspan="3">active, these compositions</td>

Pharmaceuticals may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into

213

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OF PROPERTY Λ *. INDUSTRIAL '' Nk.

<img file="MX342945B_D0398.tif" />

pharmaceutical usable preparations. The preparations formulated for oral administration can be in the form of tablets, dragees, capsules or solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture and processing the mixture of granules, after the addition of suitable auxiliaries, if desired, to obtain tablets or centers of dragees. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose (CMC) and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as, cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate.

Coated dragee cores are provided. For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,

214

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<img file="MX342945B_D0399.tif" />

polyethylene glycol (PEG) and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the coatings of tablets or dragees for the identification or characterization of different combinations of doses of active compounds.

Pharmaceutical preparations that can be used orally include hard capsules made from gelatin, as well as soft, sealed capsules made from gelatin and a plasticizer, such as glycerol or sorbitol. The hard capsules may contain the active ingredients mixed with a filler such as lactose, binders such as starches and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin or liquid polyethylene glycols (PEG). In addition, stabilizers can be added.

Depending on the particular condition or disease to be treated or prevented, additional therapeutic agents that are normally administered to treat or prevent such condition may be administered in conjunction with the inhibitors of the present invention. For example, chemotherapeutic agents or other antiproliferative agents are

215

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<img file="MX342945B_D0400.tif" />

they can combine with the inhibitors of the present invoncióa.

to treat proliferative diseases and cancer. Examples of known chemotherapeutic agents include, but are not limited to, adriamycin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, taxol, interferons, and platinum derivatives.

Other examples of non-racemic pro-oligonucleotide agents of the present invention may also be combined, including, but are not limited to, anti-inflammatory agents, such as corticosteroids, TNF blockers, IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory and immunosuppressive agents, such as, cyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, interferons, corticosteroids, cyclophophamide, azathioprine, and sulfasalazine; neurotrophic factors such as acetylcholinesterase inhibitors, MAQ inhibitors, interferons, anticonvulsants, ion channel blockers, riluzole, and anti-Parkinsonian agents; agents to treat cardiovascular disease, such as beta blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, and statins; agents to treat liver disease, such as corticosteroids, cholestyramine, interferons, and anti-viral agents; agents to treat blood disorders such as corticosteroids,

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INDUSTRIAL

<img file="MX342945B_D0401.tif" />

anti-leukemic agents and growth factors ·. agents to treat diabetes, such as, insulin, insulin analogs, alpha glucosidase inhibitors, biguanides, and insulin sensitizers, and agents to treat immunodeficiency disorders, such as, gamma globulin.

These additional agents can be administered separately, as part of a multiple dosing regimen, from the composition containing non-racemic prooligonucleotides. Alternatively, these agents can be part of a single dosage form, mixed with the non-racemic pro-oligonucleotide in a single composition.

The following examples and preparations further illustrate and exemplify the compounds of the present invention and the methods for preparing such compounds. It is understood that the scope of the present invention is in no way limited by the scope of the following examples and preparations.

EXAMPLES

Example 1: Synthesis of (SP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 5'-O- (tert-butyldiphenylsilyl) thymidine-3'-yl 3'-O- (tert-butyldimethylsilyl) thymidine-5'- The phosphorothioate [(SP) -4tt] is illustrated in Scheme A.

HO HN—&gt;

, / Ο

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INDUSTRIAL

<img file="MX342945B_D0402.tif" />

Scheme A

<td>TBOPao- ^ ha</td><td>₀nJ-O YY 0 0 BopCl</td><td></td><td>niDPBP- ^ Ha</td>

<td>Vh 'ohdbhu lt</td><td>pyridine</td><td colspan="2">OTBDPS Vf _ IT</td>

<td>'τΒΡΡ83- ^ Ha 0</td><td>you heard lt</td><td></td><td>TBOPTOyo ha</td>

<td>and L</td><td>pyridine</td><td></td><td>aW \ ^ f OTOS</td>

TIOPSO-i Λ Ba

TBDPSO-ι n Ba mcNH, 55 '0.1ΪΊ

LI pyridine or

CFrfsom

7-4-,, or

DHJ pyridine A? Y ^ οΛ&gt;

OTB S

YOUR

8-Diazabicyclo [5.4.0] undec-7-enio 5'-O- (tert-butyldiphenylsilyl) thymidine-3'-ll phosphonate (lt) (100 pmol) is dried by repeated coevaporations with dry pyridine and then dissolved in dry pyridine (1 mL). N, N '-Bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BopCl;

500 mmol) and the mixture is stirred for 5 min. A solution of amino alcohol (L-2) (100 mmol) is coevaporated several times with dry pyridine and dissolved in dry pyridine (1 mL). The amino alcohol solution is added to the reaction mixture drop by drop using a syringe and the mixture is stirred for min under argon. Dry 3-0- (tert-butyldimethylsilyl) thymidine

3t using repeated coevaporations with dry pyridine and dissolved in 100 mmol of pyridine. The mixture above

218 added through a cannula to the solution _ _

<img file="MX342945B_D0403.tif" />

butyldimethylsilyl) thymidine 3t in pyridine s

<img file="MX342945B_D0404.tif" />

After 5 min, N-trifluoroacetyl imidazole (CF3COIm; 200 mmol) is added. After an additional 30 s, add

N, Ν'-dimethylthiuram disulfide (DTD; 120 mmol). After an additional 3 min, the mixture is dried under vacuum. Concentrated NH3 (10 mL) is added to the residue and the mixture is heated for 12 h at 55 ° C. The mixture is then allowed to cool to room temperature and then concentrated to dryness under reduced pressure. The mixture is diluted with CHC13 (5 mL), and dried with

0.2 M phosphate buffer solution (pH 7.0, 5 mL). The aqueous layers are back extracted with CHC13 (5 mL, 2 times). The combined organic layers are dried over Na2SO4, filtered and concentrated to dryness under reduced pressure. The residue is purified by PTLC. The product dissolves in

CHC13 (5 mL), washed with 0.2 M buffer solution

1,8-diazabicyclo [5.4.0] undec-7-enium bicarbonate (5 mL) and back extracted with CHC13 (5 mL, 2 times). The combined organic layers are dried over Na2SO4, filtered, and concentrated to dryness to provide (SP) -4tt.

Example

The synthesis of (SP) -1,8 Diazabicyclo [5.4.0] undec-7-enio

6-N-benzoyl-5¹-O- (tert-butyldimethylsilyl) thymidine-5'-¡| phosphorothioate [(SP) -4at].

butyldif enylsilyl) -deoxydenosin-3'-yl

3'-O- (terc219

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<img file="MX342945B_D0405.tif" />

(SP) -4at is obtained from 1,8-diazabicyclo [5.4.0] undec-7-enium 6-N-benzoyl-5'-0- (tert-butyldiphenylsilyl) - deoxyadenosin-3'-yl phosphonate (la) instead of lt, using the reaction steps described in Example 1 and Scheme A for (SP) -4tt.

Example 3. Synthesis of (SP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 4-N-benzoyl-5'-O- (tert-butyldiphenylsilyl) -deoxycytidin-3'-yl 3'-O- ( tert-butyldimethylsilyl) thymidin-5'-yl phosphorothioate [(SP) -4ct].

(SP) -4ct is obtained from 1,8-diazabicyclo [5.4.0] undec-7-enium 4-N-benzoyl-5'-O- (tert-butyldiphenylsilyl) - deoxycytidin-3-yl phosphonate (le) instead of Lt, using the reaction steps described in Example 1 and Scheme A for (SP) -4tt.

Example 4. Synthesis of (SP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 2-N-phenoxyacetyl-5¹-O- (tert-butyldiphenylsilyl) -deoxyguanosine-3'-ylo 3'-O- (tert-butyldimethylsilyl) thymidine-5'-yl phosphorothioate [(SP) -4gt].

(SP) -4gt is obtained from 1,8-diazabicyclo [5.4.0] undec-7-enio 2-N-phenoxyacetyl-5'-0- (tert-butyldiphenylsilyl) - deoxyguanosin-3'-yl phosphonate (lg) in lt site, using the reaction steps described in Example 1 and Scheme A for (SP) IMPI

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OF THE MORALITY, INNUMAL OF (RP) -1.8

220

Example 5. The synthesis

Diazabicyclo [5.4.0] undec-7-enio butildif enylsilyl) thymidine-3'-ylo

<img file="MX342945B_D0406.tif" />

5'-O- (tere3'-o- (tere · butyldimethylsilyl) thymidine-5'-yl phosphorothioate [(RP) -4tt].

(RP) -4tt is produced by the transformations described in Example 1 and Scheme A for the synthesis of (SP) -4tt using amino alcohol D-2 as a chiral reagent, instead of L-2

HO \ _ tt

D-2

Example 6. Synthesis of (RP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 6-N-benzoyl-5¹-O- (tert-butyldiphenylsilyl) -deoxydenosin-3'-yl 3'-O- (tert-15-butyldimethylsilyl) thymidine-5'-yl phosphorothioate [(RP) -4at].

<td>(RP) -4at</td><td>it is produced</td><td>through</td><td>the</td><td>i transformations</td>

<td>described in the</td><td>Example 2</td><td>using</td><td>the</td><td>composed the and the</td>

<td>amino alcohol D-</td><td colspan="2">2 as a chiral reagent,</td><td>in</td><td>L-2 place.</td>

<td>Example</td><td>7. The</td><td colspan="2">synthesis</td><td>of (RP) -1,8-</td>

Diazabicyclo [5.4.0] undec-7-enium 4-N-benzoyl-5'-O- (tert-butyldiphenylsilyl) -deoxycytidin-3'-yl 3'-O- (tert-butyldimethylsilyl) thymidine-5'-yl phosphorothioate [( RP) -4ct].

(RP) -4ct is produced by the transformations

221 ^mex,cano ° E THE INDUSTRIAL PROPERTY described above in Example 3 using compound le and amino alcohol D-2 as a chiral reagent, instead of L-2.

Example 8. Synthesis of (RP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 2-N-phenoxyacetyl-5'-O- (tert-butyldiphenylsilyl) -deoxyguanosin-3'-yl 3'-O- ( tert-butyldimethylsilyl) thymidin-5'-yl phosphorothioate [(RP) -4gt].

(RP) -4gt is produced by the transformations described in Example 4 above using compound lg and amino alcohol D-2 as a chiral reagent, instead of L-2.

Scheme B

<img file="MX342945B_D0407.tif" />

<img file="MX342945B_D0408.tif" />

rtt

Example 9. Synthesis of (RP) -5'-0- (tert-butyldiphenylsilyl) thymidine-3'-yl 3'-o- (tert-butyldimethylsilyl) thymidine-5'-yl H-phosphonate [(RP) -7tt], such as it is described in Scheme B.

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<img file="MX342945B_D0409.tif" />

Lt (100 pmol) is dried by repeated coevaporations with dry pyridine and then dissolved in dry pyridine (lmL). N, N'-Bis (2-oxo-3oxazolidinyl) phosphinic chloride (BopCl; 500 pmol) is added and the mixture is stirred for 5 min. To the mixture, a solution of aminoalcohol ((aR, 2S) -6) (100 pmol) is added, which is dried by coevaporations with dry pyridine and dissolved in dry pirdine (1 mL), dropwise by syringe, and the mixture is stirred for 5 min under argon. 3'-O- (one-third butyldimethylsilyl) thymidine is dried using repeated coevaporations with dry pirdine and dissolved in 100 pmol of pyridine. The above mixture is added via cannula to the 3'-O- (tert-butyldimethylsilyl) thymidine solution

3t in dry pyridine (100 pmol). After 15 min, the mixture is concentrated under reduced pressure. The residue is diluted with CH2C12 (5 mL) and washed with saturated NaHCO3 (5 mL, 3 times). The combined aqueous layers are back extracted with CH2C12 (5 mL, 2 times). The combined organic layers are dried over Na2SO4, filtered, and concentrated to approximately 1 mL under reduced pressure. The residue is added dropwise via syringe to a 1% trifluoroacetic acid (TFA) solution in

CH2C12 dry (20 mL) at 0 ° C. After an additional 5 min, the mixture is diluted with dry CH2C12 (100 mL), and washed with aqueous solutions of saturated NaHCO3 (100 mL, 2 times). The

223

<img file="MX342945B_D0410.tif" />

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PROPERTY Combined aqueous layers are back extracted with CH2C12 ^^au(TEO^l0 times). Combined organic layers dry — dry

Na2SO4, filtered and concentrated to dryness under reduced pressure to provide crude (RP) -7tt.

Example 10. The synthesis of (RP) -6-N-benzoyl-5'-O- (tert-butyldiphenylsilyl) -deoxydenosin-3'-yl 3'-O- (tert-butyldimethylsilyl) thymidine-5'-yl H-phosphonate [(RP) - 7at].

(RP) -7at crude is produced as described in

Example 9 using la instead of lt.

Example 11. Synthesis of (RP) -4-N-benzoyl-5'-O- (tert-butyldiphenylsilyl) -deoxycytidin-3'-ylo 3'-O- (tert-butyldimethylsilyl) thymidine-5'-yl H-phosphonate [ (RP) -7ct].

(RP) -7ct gross occurs as described in

Example 9 using le instead of lt.

Example 12. Synthesis of (RP) -2-N-phenoxyacetyl-5¹-O (tert-butyldiphenylsilyl) -deoxyguanosine-3'-yl 3'-O- (tert-butyldimethylsilyl) thymidine-5'-yl H-phosphonate [(RP) -7gt].

(RP) -7gt gross is produced as described in

Example 9 using lg instead of lt.

Example 13. Synthesis of (SP) -5'-O- (tert-butyldiphenylsilyl) thymidine-3'-ylo 3'-O- (tert-butyldimethylsilyl) thymidine-5 * -yl H-phosphonate [(SP) -7tt].

(SP) -7tt gross occurs as described in

224

IMPI

MEXICAN INSTITUTE OF THE INDUSTRIAL EROP.

<img file="MX342945B_D0411.tif" />

Example 9 using (aS, 2R) -6 instead of (aR, 2S) -6 as a chiral reagent.

Example 14. Synthesis of (SP) -6-N-benzoyl-5'-O- (tert-butyldiphenylsilyl) -deoxydenosin-3'-yl 3'-O- (tert-butyldimethylsilyl) thymidine-5'-¡| H-phosphonate [(SP) -7at].

(SP) -7at gross is produced as described in

Example 9 using compound la y (aS, 2R) -6 instead of (oíR, 2S) -6 as a chiral reagent.

Example 15. Synthesis of (SP) -4-N-benzoyl-5¹-O- (tert-butyldiphenylsilyl) -deoxycytidin-3'-yl 3'-O- (tert-butyldimethylsilyl) thymidine-5'-yl H-phosphonate [(SP) -7ct].

(SP) -7ct gross occurs as described in

Example 9 using compound le y (aS, 2R) -6 instead of (aR, 2S) -6 as a chiral reagent.

Example 16. Synthesis of (SP) -2-N-phenoxyacetyl-5¹-0 (tert-butyldiphenylsilyl) -deoxyguanosine-3'-ylo 3'-O- (tert-butyldimethylsilyl) thymidine-5'-¡| H-phosphonate [(SP) -7gt].

(SP) -7gt gross is produced as described in

Example 9 using compound lg instead of lt and compound (aS, 2R) -6 instead of compound (aR, 2S) -6 as a chiral reagent.

<img file="MX342945B_D0412.tif" />

225

UJ

Example 17. Synthesis of (SP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 5-0- (tert-butyldiphenylsilyl) 2'-0- (tert-butyldimethylsilyl) uridin-3'-yl 2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(SP) -lOuu].

1,8-Diazabicyclo [5.4.0] undec-7-enium 5'-0- (tert-butyldiphenylsilyl) -2'-O- (tert-butyldimethylsilyl) uridin-3'-yl phosphonate (8u) (100 pmol) dries by repeated coevaporations with dry pyridine and then dissolved in dry pyridine (1 mL). Chloride is added

N, Ν'-Bis (2-oxo-3-oxazolidinyl) phosphinic (BopCI; 500 pmol) and the mixture is stirred for 5 min. To the mixture, a solution of aminoalcohol (L-2) (100 pmol) is added, which was dried by repeated coevaporations with dry pyridine and dissolved in dry pirdine (1 mL), drop by drop by

<img file="MX342945B_D0413.tif" />

2 6

Mexican ITUTO ..

'l PROPERTY X INDUSTRIAL X, syringe, and the mixture is stirred for 5 min under argon. It s

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridine 9u by means of dry coevaporations with dry pirdine and dissolved in 100 pmol of pyridine. Then the above mixture is added by solution of ', 3'-O-bis (third cannula butyldimethylsilyl) uridine 9u (100 pmol). After 10 min, N-trifluoroacetyl imidazole (CF3COIm; 200 pmol) is added. After an additional 30 s, N, N'-dimethylthiuram disulfide (DTD; 120 μτηοΐ) is added. After an additional 3 min, the mixture is dried under vacuum. To the residue, concentrated NH3-EtOH (3: 1, v / v, 10 mL) is added and the mixture is stirred for 12 h and then concentrated to dryness under reduced pressure. Then the mixture is diluted with CHC13 (5 mL), and dried with 0.2 M phosphate buffer (pH 7.0, 5 mL). The aqueous layers are back extracted with CHC13 (5 mL 2 times). The combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by PTLC. The product was dissolved in CHC13 (5 mL), washed with 1,820 diazabicyclo [5.4.0] undec-7-enium 0.2 M bicarbonate buffer solution (5 mL) and back extracted with CHC13 (5 mL 2 times). The combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness to provide (SP) -lOuu.

Example .

Synthesis of (SP) -1,8227

IMPI

MEXICAN INSTITUTE ΠΕ LA MONEDAD INDUSTRIAL

<img file="MX342945B_D0414.tif" />

Diazabicyclo [5.4.0] undec-7-enium 6-N-benzoyl-5'-O- (tert-butyldiphenylsilyl) -2'-O- (tert-butyldimethylsilyl) adenosin-3'-ylo

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(SP) lOau].

(SP) -lOau is produced as described in the Example using 1,8-diazabicyclo [5.4.0] undec-7-enium 6-N-benzoyl-5'O- (tert-butyldiphenylsilyl) -2'-O - (tert-butyldimethylsilyl) adenosin-3'-yl phosphonate (8a) instead of 8u.

Example 19. Synthesis of (SP) -1,810 Diazabicyclo [5.4.0] undec-7-enium 4-N-benzoyl-5¹-O- (tert-butyldipylsilyl) -2'-O- (tert-butyldimethylsilyl) cytidin-3'-yl

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-s'-yl phosphorothioate [(SP) lOcu].

(SP) -lOcu is produced as described in the Example

17 using 1,8-diazabicyclo [5.4.0] undec-7-enium 4-N-benzoyl-5'O- (tert-butyldiphenylsilyl) -2-0- (tert-butyldimethylsilyl) citidin-3'-yl phosphonate (8c) in 8u place.

Example 20. Synthesis of (SP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 2-N-phenoxyacetyl-5¹-O- (tert-20 butyldiphenylsilyl) -2-0- (tert-butyldimethylsilyl) guanosin-3'-yl

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(SP) 10Ogu].

(SP) -lOgu is produced as described in the Example

228 IMPI

MEXICAN PROPERTY INSTITUTE using 1,8-diazabicyclo [5.4.0] undec-7-ei ¥ W ™^{TO THE} phenoxyacetyl-5'-0- (tert-butyldiphenylsilyl) -2-0- (tert-butyldimethylsilyl) guanosin-3'-yl phosphonate (8g) instead of 8u.

Example 21. Synthesis of (RP) -1.85 Diazabicyclo [5.4.0] undec-7-enium 5'-O- (tert-butyldiphenylsilyl) 2'-O- (tert-butyldimethylsilyl) uridin-3'-yl 2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(RP) -lOuu].

(Rp) -lOuu is produced as described in Example 17 using a D-2 chiral reagent instead of an L-2 chiral reagent.

Example 22. Synthesis of (RP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 6-N-benzoyl-5¹-O- (tert-butyldiphenylsilyl) -2-0- (tert-butyldimethylsilyl) adenosin-3'-ylo

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(RP) 15 lOau].

(Rp) -lOau is produced as described in the Example using 8a instead of 8u and a D-2 chiral reagent instead of an L-2 chiral reagent.

Example 23. Synthesis of (RP) -1,820 Diazabicyclo [5.4.0] undec-7-enium 4-N-benzoyl-5¹-O- (tert-butyldiphenylsilyl) -2'-0- (tert-butyldimethylsilyl) citidin-3'-ylo

2 ', 3'-0-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(RP) -

<img file="MX342945B_D0415.tif" />

lOcu].

<img file="MX342945B_D0416.tif" />

₂₂₉ IMPI

INSTITUTO MEXICANO Dt LA PROPIEDAD, INOUSTRIAL, (Rp) -lOcu is produced as described in the Example using 8c instead of 8u and a D-2 chiral reagent instead of an L-2 chiral reagent.

Example 24. Synthesis of (RP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 2-N-phenoxyacetyl-5'-O- (tert-butyldiphenylsilyl) -2-0- (tert-butyldimethylsilyl) guanosin-3- the

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(RP) lOgu].

(Rp) -lOgu is produced as described in the Example using 8g instead of 8u and a D-2 chiral reagent instead of an L-2 chiral reagent.

Scheme D

TBBPSO

Ou XO OTBS H OHDflU f ~ \ ° ΓΛ

I ¿IJ or 0

ΒφΙΙ pyridine

TBDP9D w

OTBS 'Τγο-ινγ ,.

TB »

OTB DPS

OTB OPS pyridine na

<td>TBBPSOyJ » Q OTBS</td><td>^Η0γ&gt; ^³IB »OTBS Bu</td><td>TBDPSO- ^ o ^ ja OTBS</td><td>1Ϊ TFAfCMjClj</td>

<td>OR'^J'H-.</td><td>pyridine</td><td>O O'v-l n Bí λ / γ Y? V ^. · TBSO OTJS</td><td></td>

τβπρ »- ^^

H_x JO OTBS

TB »OTBS

11UU

Ba

Example 25

Synthesis of (RP) -5'-O- (tert-butyldiphenylsilyl) -2'-O- (tert-butyldimethylsilyl) uridin-3'-yl 2 ', 3'

O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(RP) -12uu].

230

IMPI

MEXICAN INSTITUTE DF. INDUSTRIAL PROPERTY

<img file="MX342945B_D0417.tif" />

It is dried 8u (100 pmol) by repeated coevaporations with dry pyridine and then dissolved in dry pyridine (lmL). N, N'-Bis (2-oxo-3oxazolidinyl) phosphinic chloride (BopCl; 500 μτηοΐ) is added and the mixture is stirred for 5 min. To the mixture, an aminoalcohol solution ((oíR, 2S) -6) (100 pmol) is added, which is dried by coevaporations with dry pyridine and dissolved in dry pirdine (1 mL), drop by syringe, and the mixture is stirred for 5 min under argon. The mixture is then added via cannula in a 9u (100 pmol) solution, which is prepared by repeated coevaporations with dry pyridine and pyridine solution. After 15 min, the mixture is concentrated under reduced pressure. The residue is diluted with CH2C12 (5 mL) and washed with saturated NaHCO3 (5 mL 3 times). The combined aqueous layers are back extracted with CH2C12 (5 mL 2 times). The combined organic layers were dried over

Na2SO4, filtered and concentrated to about 1 mL under reduced pressure. The residue is added dropwise via syringe to a solution of 1% trifluoroacetic acid (TFA) in dry CH2C12 (20 mL) at 0 ° C. After an additional 5 min, the mixture is diluted with dry CH2C12 (100 mL), and washed with aqueous solutions of saturated NaHCO3 (100 mL 2 times). The combined aqueous layers are back extracted with CH2C12 (100 mL 2 times). The combined organic layers are

231

ΙΜΡΙ

<img file="MX342945B_D0418.tif" />

Dried over Na2SO4, filtered and concentrated to dryness under reduced pressure to provide crude (Rp) -12uu, which is analyzed by 31P NMR.

Example 26. Synthesis of (RP) -6-N-benzoyl-5'-O- (tert-5-butyldiphenylsilyl) -2'-0- (tert-butyldimethylsilyl) adenosin-3'-ylo

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(RP) 12au].

(RP) -12au gross occurs as described in

Example 25 using 8a instead of 8u.

Example 27. Synthesis of (RP) -4-N-benzoyl-5'-O- (tert-butyldiphenylsilyl) -2'-O- (tert-butyldimethylsilyl) cytidin-3'-ylo

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(RP) -12 cu].

(RP) -12cu crude is produced as described in

Example 25 using 8c instead of 8u.

Example 28. Synthesis of (RP) -2-N-phenoxyacetyl-5'-0 (tert-butyldiphenylsilyl) -2'-0- (tert-butyldimethylsilyl) guanosin-3'-ylo

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(RP) -12gu].

(RP) -12gu gross is produced as described in

Example 25 using 8g instead of 8u.

Example 29. Synthesis of (SP) -5¹-O- (tert-butyldiphenylsilyl) -2-0- (tert-butyldimethylsilyl) uridin-3'-yl 2 ', 3'

232

IMPI 'NSTITUTO MEXICANO CE THE INDUSTRIAL PROPERTY

<img file="MX342945B_D0419.tif" />

O-bis (tert-butyldimethylsilyl) uridin-5'-ll H-phosphonate [(SP) -12uu].

Crude (SP) -12uu is produced as described in Example 25 using a chiral reagent (aS, 2R) -6 instead of a chiral reagent (oIR, 2S) -6.

Example 30. Synthesis of (sP) -6-N-benzoyl-5¹-O- (tert-butyldiphenylsilyl) -2-0- (tert-butyldimethylsilyl) adenosin-3'-yl

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(SP) 12au].

(SP) -12au gross occurs as described in

Example 25 using 8a instead of 8u and a chiral reagent (aS,

2R) -6 instead of a chiral reagent (oIR, 2S) -6.

Example 31. Synthesis of (SP) -4-N-benzoyl-5'-0- (tert-butyldipylsilyl) -2'-0- (tert-butyldimethylsilyl) cytidin-3'-yl

2 ', 3'-0-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(SP) 15 12cu].

(SP) -12au gross occurs as described in

Example 25 using 8c instead of 8u and a chiral reagent (aS,

2R) -6 instead of a chiral reagent (aR, 2S) -6.

Example 32. Synthesis of (SP) -2-N-phenoxyacetyl-5'-O20 (tert-butyldiphenylsilyl) -2'-O- (tert-butyldimethylsilyl) guanosin-3'-yl 2 ', 3'-O-bis (tert-butyldimethylsilyl ) uridin-5'-yl H-phosphonate [(SP) -12gu].

(SP) -12gu gross is produced as described in

233 ΙΜΡΙ

MEXICAN INSTITUTE

OF THE PROPERTY

Example 25 using 8g instead of 8u and a reagent

<img file="MX342945B_D0420.tif" />

2R) -6 instead of a chiral reagent (aR, 2S7-6.

Scheme E

TBDP30 or or

V_7 BBp! Lt \ 5 / V HO HN--,

ΎίΥ, Λ &lt;'

L4

TBSO Ov_p^ D HDBU * O 'h

13U pyridine pyridine ^Η° γ&gt; γ^ΒίTBSD OR IB a

Its pyridine

CFtfom στο /

NH

BJiENHa-etoH 0: 1, «*) rt 12 I

TBQPSO

-, _Λ B nao pyridine ⁰

RATE 0TB8 1 &lt;UU

Example

Synthesis of (SP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 5'-O- (tert-butyldiphenylsilyl) 3'-O- (tert-butyldimethylsilyl) uridin-2'-yl 2 ', 3' -O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(SP) -14uu].

1,8-Diazabicyclo [5.4.0] undec-7-enio 5'-O- (tert-butyldipylsilyl) -3-0- (tert-butyldimethylsilyl) uridin-3'-yl phosphonate (13u) (100 pmol) dries by repeated coevaporations with dry pyridine and then dissolved in dry pyridine (1 mL). N, N'-B¡s (2-oxo-3oxazolidinyl) phosphinic chloride (BopCl; 500 pmol) is added and the mixture is stirred for 5 min. To the mixture, a solution of aminoalcohol (L-2) (100 pmol) is added, which was dried by repeated coevaporations with dry pyridine and dissolved in dry pirdine (1 mL), dropwise by syringe, and the

234

IMPI Mexican Institute of industrial PROPERTY

<img file="MX342945B_D0421.tif" />

The mixture is stirred for 5 min under argon. 2 ', 3'-Qbis (tert-butyldimethylsilyl) uridine 9u is dried by repeated coevaporations with dry pirdine and dissolved in 100 pyridine pinol. Then the above mixture is added by cannula into the solution of 2 ', 3'-O-bis (tert-butyldimethylsilyl) uridine 9u (100 μπιοί). After 10 min, N-trifluoroacetyl imidazole (CF3COIm; 200 μιηοΐ) is added. After an additional 30 s, N, N'-dimethylthiuram disulfide (DTD; 12 0 μπιοί) is added. After an additional 3 min, the mixture is dried under vacuum. To the residue, concentrated NH3-EtOH (3: 1, v / v, 10 mL) is added and the mixture is stirred for 12 h and then concentrated to dryness under reduced pressure. Then the mixture is diluted with CHC13 (5 mL), and dried with 0.2M phosphate buffer (pH 7.0, 5 mL). The aqueous layers are back extracted with CHC13 (5 mL 2 times). The combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by PTLC. The product was dissolved in CHC13 (5 mL), washed with a buffer of 1,820 diazabicyclo [5.4.0] undec-7-enium 0.2M bicarbonate (5 mL) and back extracted with CHC13 (5 mL 2 times). The combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness to provide (SP) -14uu.

.

Example

Synthesis of (SP) -1.8235

Diazabicyclo [5.4.0] undec-7-enio

IMPI

MRXICANO INSTITUTE OF INDUSTRIAL PROPERTY

6-N-benzoyl-5'-O- (tert-

<img file="MX342945B_D0422.tif" />

butyldiphenylsilyl) -3'-0- (tert-butyldimethylsilyl) adenosin-2-yl

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(SP) 14au].

(SP) -14au is produced as described in the Example using 1,8-diazabicyclo [5.4.0] undec-7-enium 6-N-benzoyl-5'O- (tert-butyldiphenylsilyl) -3-0- (tert-butyldimethylsilyl) adenosin-2'-yl phosphonate (13a) instead of 13u.

Example 35. Synthesis of (SP) -l, ΒΙΟ Diazabicyclo [5.4.0] undec-7-enium 4-N-benzoyl-5¹-O- (tert-butyldiphenylsilyl) -3-0- (tert-butyldimethylsilyl) cytidin-2'-yl

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(SP) 14cu].

(SP) -14cu is produced as described in the Example

33 using 1,8-diazabicyclo [5.4.0] undec-7-enium 4-N-benzoyl-5'O- (tert-butyldiphenylsilyl) -3'-0- (tert-butyldimethylsilyl) citidin-2'-yl phosphonate (13c) instead of 13u.

Example 36. Synthesis of (SP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 2-N-phenoxyacetyl-5'-O- (tert-20 butyldiphenylsilyl) -3'-O- (tert-butyldimethylsilyl) guanosin- 2'-yl

2 ', 3'-0-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(SP) 14gu].

(SP) -14gu is produced as described in the Example

IMPI

3 6 MEXICAN INSTITUTE

Dt THE PROPERTY

INDUSTRIAL using 1,8-diazabicyclo [5.4.0] undec-7-enium

<img file="MX342945B_D0423.tif" />

2-Nphenoxy acetyl-5'-0- (tert-butyldiphenylsilyl) -3'-O- (tert-butyldimethylsilyl) guanosin-2'-yl phosphonate (13g) instead of 13u.

Example 37. Synthesis of (RP) -1.85 Diazabicyclo [5.4.0] undec-7-enium 5'-O- (tert-butyldifynylsilyl) 3'-O- (tert-butyldimethylsilyl) uridin-2'-yl 2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(RP) -14uu].

(Rp) -14uu is produced as described in Example 33 using a D-2 chiral reagent instead of an L-2 chiral reagent.

Example 38. Synthesis of (RP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 6-N-benzoyl-5¹-O- (tert-butyldipylsilyl) -3-0- (tert-butyldimethylsilyl) adenos in-2'-yl

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(RP) 15 14au].

(Rp) -14au is produced as described in the Example using 13a instead of 13u and a D-2 chiral reagent instead of an L-2 chiral reagent.

Example 39. Synthesis of (RP) -1,820 Diazabicyclo [5.4.0] undec-7-enium 4-N-benzoyl-5¹-O- (tert-butyldipylsilyl) -3-0- (tert-butyldimethylsilyl) citidin-2'-yl

2 ', 3'-o-bis (tert-butyldimethylsilyl) uridin-5'-ll phosphorothioate [(RP) 14cu].

237

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0424.tif" />

(Rp) -14cu is produced as described in the Example using 13c instead of 13u and a D-2 chiral reagent instead of an L-2 chiral reagent.

Example 40. Synthesis of (RP) -1,8 Diazabicyclo [5.4.0] undec-7-enium 2-N-phenoxyacetyl-5¹-O- (tert-butyldipylsilyl) -3'-0- (tert-butyldimethylsilyl) guanos in-2-yl

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl phosphorothioate [(RP) 14gu].

(Rp) -14gu is produced as described in the Example using 13g instead of 13u and a D-2 chiral reagent instead of an L-2 chiral reagent.

Esauema F

TBDPSO- ^ o ^ a

TBSO 0 ^ -0 HDBlh-O Ή

13u

Γ ~ λ? Γ ~ \

YlíY oo BopCI pyridine

HO HN-, «« .as) * ^HOI- ^^to

TBSO OTBS

9u pyridine pyridine

<img file="MX342945B_D0425.tif" />

ISuu (RP) -5¹-O- (tert Example 41. Synthesis butyldiphenylsilyl) -3'-0- (tert-butyldimethylsilyl) uridin-2'-yl 2 ', 3'

O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(RP) -15uu].

of

Dried 13u (100 pmol) by coevaporation

238

IMPI

MEXICAN PROPERTY INSTALLER

INDUSTRIAL

<img file="MX342945B_D0426.tif" />

repeated with dry pyridine and then dissolved in dry pyridine (lmL). N, N'-Bis (2-oxo-3oxazolidinyl) phosphinic chloride (BopCl; 500 pmol) is added and the mixture is stirred for 5 min. To the mixture, an aminoalcohol solution ((aR, 2S) -6) (100 pmol) is added, which is dried by coevaporations with dry pyridine and dissolved in dry pirdine (1 mL), drop by syringe, and the mixture is stirred for 5 min under argon. The mixture is then added via cannula in a 9u (100 pmol) solution, which is prepared by repeated coevaporations with dry pyridine and pyridine solution. After 15 min, the mixture is concentrated under reduced pressure. The residue is diluted with CH2C12 (5 mL) and washed with saturated NaHCO3 (5 mL 3 times). The combined aqueous layers are back extracted with CH2C12 (5 mL 2 times). The combined organic layers were dried over Na2SO4, filtered, and concentrated to approximately 1 mL under reduced pressure. The residue is added dropwise via syringe to a solution of 1% trifluoroacetic acid (TFA) in dry CH2C12 (20 mL) at 0 ° C. After an additional 5 min, the mixture is diluted with dry CH2C12 (100 mL), and washed with aqueous solutions of saturated NaHCO3 (100 mL 2 times). The combined aqueous layers are back extracted with

CH2C12 (100 mL 2 times). The combined organic layers were dried over Na2SO4, filtered, and concentrated to

239

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0427.tif" />

Drying under reduced pressure to provide crude (Rp) -15uu, which is analyzed by 31P NMR.

Example 42. Synthesis of (RP) -6-N-benzoyl-5'-0- (tert-butyldiphenylsilyl) -3'-0- (tert-butyldimethylsilyl) adenosin-2'-yl

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(RP) 15au].

(RP) -15au gross occurs as described in

Example 41 using 13a instead of 13u.

Example 43. Synthesis of (RP) -4-N-benzoyl-5¹-0- (third butyldiphenylsilyl) -3'-0- (tert-butyldimethylsilyl) cytidin-2'-yl

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(RP) -15 cu].

(RP) -15cu gross is produced as described in

Example 41 using 13c instead of 13u.

Example 44. Synthesis of (RP) -2-N-phenoxyacetyl-5¹-0 (tert-butyldiphenylsilyl) -3'-0- (tert-butyldimethylsilyl) guanosin-2'-ylo 2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(RP) - 15gu].

(RP) -15gu gross is produced as described in

Example 41 using 13g instead of 13u.

Example 45. Synthesis of (SP) -5¹-O- (tert-butyldiphenylsilyl) -3-0- (tert-butyldimethylsilyl) uridin-2'-ylo 2 ', 3'O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(SP) - 15uu].

240

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342945B_D0428.tif" />

Crude (SP) -15uu is produced as described in Example 41 using a chiral reagent (aS, 2R) -6 instead of a chiral reagent (oir, 2S) -6.

Example 46. Synthesis of (SP) -6-N-benzoyl-5¹-O- (tert-butyldipylsilyl) -3'-0- (tert-butyldimethylsilyl) adenos in-2-yl

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(SP) 15au].

(SP) -15au gross occurs as described in

Example 41 using 13a instead of 13u and a chiral reagent (aS, 2R) -6 instead of a chiral reagent (oIR, 2S) -6.

Example 47. Synthesis of (SP) -4-N-benzoyl-5¹-O- (tert-butyldipylsilyl) -3'-O- (tert-butyldimethylsilyl) citidin-2'-ylo

2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(SP) 15cu].

(SP) -15cu gross is produced as described in

Example 41 using 13c instead of 13u and a chiral reagent (aS, 2R) -6 instead of a chiral reagent (aR, 2S) -6.

Example 48. Synthesis of (SP) -2-N-phenoxyacetyl-5¹-0 (tert-butyldif enylsilyl) -3'-O- (tert20 butyldimethylsilyl) guanosin-2'-ylo 2 ', 3'-O-bis (tert-butyldimethylsilyl) uridin-5'-yl H-phosphonate [(SP ) -15gu].

Crude (SP) -15gu is produced as described in Example 41 using 13g instead of 13u and a chiral reagent

IMPI

MEXICAN INSTITUTE

OE THE PROPERTY

INDUSTRIAL

2S) -6.

241 (aS, 2R) -6 instead of a chiral reagent (aR,

<img file="MX342945B_D0429.tif" />

Scheme G: Synthesis of nucleic acid prodrugs of

S-acyl-2-thioethyl.

<img file="MX342945B_D0430.tif" />

NCB HO '' ''³® ^βΊ^βPyridine

<img file="MX342945B_D0431.tif" />

OTBS

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Example 49. Synthesis of S-acyl-2-thioethyl nucleic acid prodrug of (RP) -timidin-3'-yl thymidin-5'-yl phosphonate [(RP) -16tt] as described in Scheme G .

(RP) -5 '-O- (tert-Butyldiphenylsilyl) thymidin-3'-yl 3'-O- (tert-15 butyldimethylsilyl) timdin-5'-yl H-phosphonate [(RP) -7tt] (100 pmol) se Dries by repeated coevaporations with dry pyridine and then dissolves in dry pyridine (1 mL). N-Chlorosuccinimide (0.1 mmol) is added and the mixture is stirred for 2 hours at 0 ° C. The mixture is concentrated and dissolved in dry pyridine (1 mL). The above mixture is treated with S-acetyl-2-thioethanol (100 pmol) in dry pyridine (100 pmol). After 1 hour, the mixture is concentrated and then dissolved in triethylamine trihydrofluoride (500 pL). The mixture is stirred for 15 h at room temperature. Then it is added to the

242

IMPI

MEXICAN INSTITUTE OF THE KF.OPIEDAP INDUSTRIAL

<img file="MX342945B_D0433.tif" />

mix a buffer solution of ammonium acetate 0.

(2.5 mL) and the mixture is washed with Et2O (3 mL 3 times). The combined organic layers were back extracted with 0.1M ammonium acetate buffer (3 mL). The combined aqueous layers were then concentrated to dryness under reduced pressure and the residue was purified by reversed phase column chromatography [a linear gradient of 0-10% acetonitrile in 0.1M ammonium acetate buffer (pH 7.0)] to provide (RP) -16tt.

Example 50. Synthesis of S-acyl-2-thioethyl pronucleotide from (RP) -6-N-benzoyl-deoxyadenosin-3'-yl thymidin-5'-yl phosphonate [(RP) -16at].

(RP) -16at gross is produced as described in

Example 49 using (RP) -7at instead of (RP) -7tt.

Example 51. Synthesis of S-acyl-2-thioethyl pronucleotide from (RP) -4-N-benzoyl-deoxycytidine-3'-yl thymidine-5'-yl phosphonate [(RP) -16ct].

(RP) -16ct gross occurs as described in

Example 49 using (RP) -7ct instead of (RP) -7tt.

Example 52. Synthesis of S-acyl-2-thioethyl pronucleotide from (RP) -2-N-phenoxyacetyl-deoxyguanosin-3'-yl thymidin5'-yl phosphonate [(RP) -16gt].

(RP) -16gt gross is produced as described in

243

ΙΜΡΙ

MEXICAN INSTITUTE. -. -, ν,, Dt THE PROPERTY

Example 49 using (RP) -7gt instead of (RP) -7tt. industrial

<img file="MX342945B_D0434.tif" />

Example 53. Synthesis of pronuc leot id ~~~ of 0 "aei 1" 2 thioethyl of (SP) -timidin-3'-lo t¡midin-5'-il phosphonate [(RP) -16tt].

(SP) -16tt gross is produced as described in

Example 49 using (SP) -7tt instead of (RP) -7tt.

Example 54. Synthesis of S-acyl-2-thioethyl pronucleotide from (SP) -6-N-benzoyl-deoxyadenosin-3'-ylo tmidmid-5'-yl phosphonate [(SP) -16at].

(SP) -16at gross is produced as described in

Example 49 using (SP) -7at instead of (RP) -7tt.

Example 55. Synthesis of S-acyl-2-thioethyl pronucleotide from (SP) -4-N-benzoyl-deoxycytidine-3'-yl tmididin-5'-yl phosphonate [(SP) -16ct].

(SP) -16ct gross occurs as described in

Example 49 using (SP) -7ct instead of (RP) -7tt.

Example 56. Synthesis of S-acyl-2-thioethyl pronucleotide from (SP) -2-N-phenoxyacetyl-deoxyguanosin-3'-yl thymidine 5'-y phosphonate [(SP) -16gt].

Crude (SP) -16gt is produced as described in Example 49 using (SP) -7gt instead of (RP) -7tt.

Example 57. Synthesis of S-acyl-2-thioethyl pronucleotide from (RP) -uridin-3'-ylo uridin-5'-yl phosphonate [(RP) -16uu].

(RP) -16uu gross occurs as described in