Heterobifunctional linkers with polyethylene glycol segments and immune response modifier conjugates made therefrom.

(54) Title: HETEROBIFUNCIONAL BINDERS WITH POLYETHYLENE GLYCOL SEGMENTS AND IMMUNE RESPONSE-MODIFYING CONJUGATES MADE FROM THEM.

(54) Title: HETEROBIFUNCTIONAL LINKERS WITH POLYETHYLENE GLYCOL SEGMENTS AND IMMUNE RESPONSE MODIFIER CONJUGATES MADE THEREFROM.

(57) Summary

The present invention relates to conjugates of an immune response modifier, a linker, and an antigen. The linker is represented by the formula (I): where A is CH or N, p is in a range of 1 to 50, R is a bond or -alkylene-O-, R 'is alkylene which is optionally interrupted or terminated with one or more amide or ether groups, and E is an amine or thiol reactive group. Also disclosed are pharmaceutical compositions containing the compound or conjugate, methods of making a conjugate, and methods of using the compounds or conjugates as immunomodulators to induce cytokine biosynthesis in an animal and to vaccinate an animal. A linker modified antigen is also described.

(57) Abstract

Conjugates of an immune response modifier, a linker, and an antigen are disclosed. The linker is represented by formula (I): wherein A is CH or N, p is in a range from 1 to 50, R is a bond or -alkylene-O-, R 'is alkylene that is optionally interrupted or terminated with one or more amide or ether groups, and E is an amine- or thiol-reactive group. Pharmaceutical compositions containing the compound or the conjugate, methods of making a conjugate, and methods of use of the compounds or conjugates as immunomodulators for inducing cytokine biosynthesis in an animal and for vaccinating an animal are also disclosed. An antigen modified by the linker is also disclosed.

Mexican Institute of Industrial Property

<img file="MX347240B_D0001.tif" />

PATENT TITLE NO. 347240

Headlines):

Home

3M INNOVATIVE PROPERTIES COMPANY

3M Center, Saint Paul, Minnesota, 55144-1000, USA

Denomination:

LIGATORS

HETEROBIFUNCTIONALS

WITH

SEGMENTS

POLYETHYLENE GLYCOL AND IMMUNE RESPONSE MODIFYING CONJUGATES MADE FROM THE SAME.

Classification:

Inventor (s):

Int.CI.8: A61K47 / 48: C07C233 / 69; C07D207 / 46: C07D213 / 82: C07D249 / 18; C07D471 / 04

PAUL D. WIGHTMAN

REQUEST

Number:

International filing date!

MX / a / 2013/014146 June 2012

PRIORITY

Country:

Date:

Number:

US US June 2011 June 2011

61/493,143

61/493,051

Validity: Twenty years

Expiration Date: June 1, 2032

The reference patent is granted on the basis of articles 1^or, 2 'fraction V, 6^or Section III, and 59 of the Industrial Property Law.

In accordance with article 23 of the Industrial Property Law, this patent is valid for twenty years, non-extendable, counted from the filing date 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 on Industrial Property grounds (Official Gazette of the Federation (DOF

Sections III and 7 bis 2 of Law 994, 10/25/1996, 12/26/1987, 05/17/1999,

01/26/2004, 06/16/2005, 01/25/2006, 05/06/2009, 06/01/2010, 06/18/2010; «W06 / 2O1O, 01/27/2012 and 04/09/2012); articles 1 '3^or fraction V. clause a), 4^or and 12th fractions i and m of the Regulations of the Mexican Institute of Industrial Property (DOF 12/14/1998, amended on 07/01/2002,15/07/2004, 07/28/2004 and 09/07/2007); Articles 1, 3, 4, 5, section V, subsection a), 16 sections I and m and 30 tfeTB Organic Matute 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), 1st, 3rd and 5th subsection a) of the Agreement that delegates powers to the Deputy General Directors, Coordinator, Divisional Directors, Heads of 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="MX347240B_D0002.tif" />

Issue Date: April 20, 2017

THE DIVISIONAL DIRECTOR OF PATENTS

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NAHANNY CANAL REYES

MX / 2017/33788

3ΥΜ0

<img file="MX347240B_D0004.tif" />

HETEROBIFUNCTIONAL BINDERS WITH POLYETHYLENE GLYCOL SEGMENTS

AND CONJUGATES MODIFYING THE IMMUNE RESPONSE

MADE FROM THEM

Background of the Invention

In recent years there has been an effort, with significant success, to discover new pharmacological compounds that act by stimulating certain key aspects of the immune system, as well as by suppressing certain other aspects (see, for example, United States Patent Nos. 6,039,969 (Tomai et al.) And 6,200,592 (Tomai et al.). These compounds, referred to herein as immune response modifiers (IRMs), appear to act through basic mechanisms of the immune system known as toll-like receptors (TLRs) to induce Selected cytokine biosynthesis, induction of costimulatory molecules, and increased antigen presentation capacity.

Many MRIs can be helpful in treating a wide variety of disorders and conditions. For example, certain MRIs may be useful in treating viral disorders (eg, human papillomavirus, hepatitis, herpes), neoplasms (eg, basal cell carcinoma, squamous cell carcinoma, actinic keratosis,

Ref. 245438

IMPI ^

MEXICAN INSTITUTE

OF INDUSTRIAL PROPERTY OiaJíl&gt; 3 * ”melanoma), disorders mediated by T_H2 (eg, asthma, allergic rhinitis, atopic dermatitis), and autoimmune disorders.

Many known MRIs are imidazoquinoline amine derivatives (see, for example, US Patent No. 4,689,338 (Gerster)), but other classes of compounds are also known (see, for example, US Patent Nos. 5,446,153 (Lindstrom et al.); 6,194,425 (Gerster et al.); And 6,110,929 (Gerster et al.); And International Publication Number WO2005 / 079195 (Hays et al.)) While more are still being discovered.

Certain MRIs may also be useful, for example, as vaccine adjuvants. In some cases, an MRI compound can be administered in a conjugated composition in which the IRM compound is covalently linked to an antigenic moiety (see, for example, US Patent No. 7,427,629 (Kedl et al.) And Application for United States Patent Publication No. 2009/0035323 (Stoermer et al.)).

In view of the great therapeutic potential for MRIs in treating a wide variety of disorders and conditions, and despite the significant work that has already been done, there is still a need for expanded uses, compositions, and administration options for MRI compounds. .

ΙΜΡΙ (

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

Brief Description of the Invention

<img file="MX347240B_D0005.tif" />

.... -----— V

The present invention provides new conjugates that include an immune portion (IRM). New ones for example, to generate response modifier conjugates may be useful, an antigen-specific immune response. In one aspect, the present invention provides a conjugate comprising a reaction product of a hydrazine- or hydrazide-substituted immune response modifier; a linker represented by the formula:

(/ Λ J ^ c (0) -nh-r '' - (ch₂ch₂0) ^ - e a.

where A, p, R ', R, and E are as defined below; and an antigen.

In yet another aspect, the present invention provides a conjugate comprising an immune response modifier; a linker represented by the formula:

* / —C (O&gt; NH-R »(CH₂CH_?O) -R - ** A—- ¹ where A, p, R ', and R are as defined below; and an antigen; wherein the immune response modifier is covalently linked to the linker at * through a hydrazone functional group, and wherein the antigen is covalently linked to the linker at ** through ______

<img file="MX347240B_D0006.tif" />

of an amide, disulfide, urea, thiourea, carbamate, or a carbon-sulfur or carbon-nitrogen bond in the alpha position to an amide or sulfone or directly linked to a succinimide ring.

In yet another aspect, the present disclosure provides a method of making a conjugate, the method comprises combining an antigen with a linker to provide a modified antigen, wherein the linker is represented by the formula:

<img file="MX347240B_D0007.tif" />

where A, p, R¹, R, and E are as defined below; and combining the modified antigen with a hydrazine or hydrazide substituted immune response modifier to provide the conjugate.

The conjugates of the present invention can induce cytokine biosynthesis (eg, induce the synthesis of at least one cytokine) and otherwise modulate the immune response when administered to animals. The ability to induce cytokine biosynthesis in animals makes the conjugates useful in the treatment of a variety of conditions such as viral disorders and tumors that respond to such changes in response.

IMPI ^^

MEXICAN INSTITUTE

DI THE PROPERTY 'CeewPítfF INDUSTRIAL ^ * _ YES— Immune. Consequently, the present invention __1 -.- 11 ^ 1 —II »¹ IT: --- i · provides a method of inducing cytokine biosynthesis in an animal, by administering to the animal an effective amount of a conjugate described herein.

Co-administration of a vaccine adjuvant (for example, an MRI compound such as a compound of Formula I or II described below) and an antigen to an immune cell can increase the immune response to the antigen and improve immune memory. antigen specific. Optimal distribution can occur, for example, when the adjuvant and the antigen are processed within an antigen-presenting cell at the same time, for example, when they are covalently linked as in the conjugates of the present invention. Accordingly, the present invention further provides a method of vaccinating an animal, comprising administering to the animal a conjugate described herein.

The invention further provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a conjugate described herein.

Advantageously, the conjugates according to the present invention can be prepared under conditions that do not denature the antigens (for example, which can be proteins). For example, conjugates can be

<img file="MX347240B_D0008.tif" />

prepared at physiological pH. Also, coval links ·⁰* · ^ Formed to make conjugates do not require irradiation. The linker used to make the conjugates is advantageous, for example, to promote the solubility and stability of the antigen (eg, which in some embodiments is a protein). Accordingly, in certain embodiments , the present invention further provides a compound represented by the formula:

. fg '~ fc · 1 / X £ ·'

A where A, p, and LG are as defined below; and a modified antigen that has at least one segment represented by the formula:

Vc (O) -NH- (CH₂CH₂O) -CH₂CH₂-C (O) -N * (H) A *

where A and p are as defined below, and the nitrogen atom indicated by N * is covalently bound to the antigen.

Also advantageously, in many embodiments, including those embodiments where the hydrazine or hydrazide substituted immune response modifier comprises an aromatic ring to which the hydrazine or hydrazide group is attached, the formation of the conjugate can be readily monitored using spectroscopy.

<img file="MX347240B_D0009.tif" />

<img file="MX347240B_D0010.tif" />

INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL de UV due to the characteristic absorption of the hydrazone bond that is formed.

The terms "comprise" and variations thereof, do not have a limiting meaning where these terms appear in the description and in the claims.

As used herein, a, an, a, the, the, at least one, and one or more are used interchangeably.

Also herein, indications of numerical ranges by endpoints include all numbers encompassed within this range (for example, 1 through 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.) .

Antigen refers to any substance that can be bound by an antibody in a manner that is immunospecific to some degree for a humoral immune response. Antigen as used herein also refers to any substance that can be bound by an antigen presenting cell for a cell-mediated immune response. An antigen described herein can promote antigenic activity including, for example, one or more of the following: generation of antibodies specific for the antigen by B cells, maturation of immune cells,

<img file="MX347240B_D0011.tif" />

⁸ IMPI

INSTITUTO MEXICANO DE LA PROPERTY INDUSTRIAL production of cytokines by immune cells, and the generation of antigen-presenting cells that present the antigen. Antigens useful for practicing the present disclosure include those that have very weak activity and / or have no therapeutic benefit in the absence of an adjuvant (eg, such as an MRI compound).

A conjugate as used herein is a compound that contains two components (eg, an IRM compound and an antigen) covalently linked to each other.

Induce and variations thereof refers to any measurable increase in cellular activity. For example, the induction of an immune response may include, for example, an increase in the production of a cytokine, the activation, proliferation, or maturation of a population of immune cells, and / or other indicators of increased immune function.

The term protein includes proteins and glycoproteins. For protein antigens, modifications can be made to a particular antigen without rendering the modified antigen unsuitable for use as an antigen. For example, one or more portions of the amino acid sequence of a protein antigen can be deleted or substituted or amino acids can be added.

IMPI 'NSTrruro Mexicano μ U PROHiDAD

INDUSTRIAL

<img file="MX347240B_D0012.tif" />

additional, and the protein antigen can still retain antigenic activity.

The term hydrazine refers to a functional group of the formula -NHNH₂.

The term hydrazide refers to a functional group of the formula -C (0) NHNH₂.

The term "hydrazone" refers to a functional group of the formula -NHN = C (R) - or -C (0) NHN = C (R) -, where

R is hydrogen or alkyl, for example.

The above brief description of the present invention is not intended to describe every disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies the illustrative embodiments. At various sites throughout the description, guidance is provided through lists of examples, the examples of which can be used in various combinations. In each case, the indicated list serves only as a representative group and should not be construed as an exclusive list.

Detailed description of the invention

In one embodiment, the present invention provides a conjugate comprising a reaction product of:

a hydrazine or hydrazide substituted immune response modifier;

<img file="MX347240B_D0013.tif" />

IMPI

INSTITUTO MEXICANO DE LA FROFIEDAD INDUSTRIAL a linker represented by the formula:

or \\ / -C (O) -NH-R- (CH₇CH₂OR)_p-RE

A— '~ where A is CH or N, p is in a range of 1 to 50, R is a bond or -alkylene-O-, R' is alkylene which is optionally interrupted or terminated with one or more amide groups or ether, and E is an amine or thiol reactive group; and an antigen.

In one embodiment, the present invention provides a conjugate comprising:

an immune response modifier, a linker represented by the formula:

* '^ / - aO) -NH-R ”- (CH₂CH, O)_p-R'- · *

TO-⁷ "Where A is CH or N, p is in a range from 1 to

R is an o -alkylene-O- bond, and R 'is alkylene which is optionally interrupted or terminated with one or more amide or ether groups; and an antigen;

wherein the immune response modifier is covalently linked to the linker at * through a hydrazone functional group, and wherein the antigen is covalently linked to the linker at **.

The present invention further provides a method of making a conjugate, the method comprises: combining

<img file="MX347240B_D0014.tif" />

IMPI

INSTITUTO MEXICANO DE IA FMMSDAD INDUSTRIAL an antigen with a linker to provide a modified antigen, where the linker is represented by the formula:

<img file="MX347240B_D0015.tif" />

where A is CH or N, p is in a range from 1 to

50, R is a bond or

-alkylene-O-, R 'is alkylene which is optionally interrupted or terminated with one or more amide or ether groups, and E is an amine or thiol reactive group;

and combining the modified antigen with a hydrazine or hydrazide substituted immune response modifier to provide the conjugate.

For any of the conjugates presented herein, each of the following variables (for example, A, p R ', E, R2, R3, X, Y, n, and so on) in any of its modalities, can be combined with one or more of the other variables in any of their embodiments and associated with any of the formulas or IRM compounds described herein, as might be understood by one of ordinary skill in the art. Each of the resulting combinations of the variables is a modality of the

<img file="MX347240B_D0016.tif" />

In some modes, A is CH or N. In some

IMPI

INSTITUTO MIXICANO DE LA RROHIDad INDUSTRIAL present invention.

modalities, A is CH.

In some modalities, including any of the previous modalities of the conjugates where A is defined, p is e: modalities, p is modalities, p is modalities, p is modalities, p is modalities, p is modalities, p is modalities, p is modalities, p is modalities, p is modalities, p is modalities, p is an interval of 1 in a interval of in an interval of in an interval of in an interval of in an interval of in a interval of in an interval of in an interval of in an interval of: n a range of 4 to 50. In some a 50. In some to 40. In some to 40. In some to 30. In some to 30. In some to 24. In some to 16. In some to 12. In some to 24. In some to 16. In some to 12.

In some modalities, including any of the previous modalities of the conjugates where A or op are defined, R¹ alkylene that is optionally interrupted or terminated with one or more amide or ether groups.

In some of these embodiments, R 'is ethylene. In some of these embodiments, R 'is propylene. In some modalities, R¹ it is alkylene that is interrupted by one or two amide groups.

<img file="MX347240B_D0017.tif" />

IMPI

INSTITUTO MEXICANO OF. INDUSTRIAL PROPERTY

In some embodiments, they include any of the above embodiments of the conjugates where A, p, or R 'are defined, R is a bond or -alkylene-O-. In some embodiments, R is a link. In these embodiments, it will be understood that R could be absent from the structural formula of the linker. In some embodiments, R is propylene-0-.

In some embodiments, including any of the above embodiments of conjugates where A, p, R ', or R are defined, E is an amine or thiol reactive group. Suitable amine or thiol reactive groups include maleimide, vinyl sulfone, acrylamide, pyridyldisulfide, methylsulfonyldisulfide, N-hydroxysuccinimide ester, sulfo-Nhydroxysuccinimide ester or a salt thereof, 4nitrophenyl ester, acid bromide, acid, acid anhydride, pentafluorophenyl ester, tetrafluorophenyl ester, N-hydroxybenzotriazole ester, acetyl iodine, bromoacetyl, chloroacetyl, succinimidyl carbonate, chloroformate, -0C (0) -O-CH (C1) CC1₃ , 0C (0) -0- (4-nitrophenyl), isocyanate, and thioisocyanate groups.

In some embodiments, including any of the above embodiments of conjugates where A, p, R ', or R are defined, Ξ is an ester selected from

INSTITUTO MEXICANO DE LA PROPIEDAD INDUSTRIAL ------- ► group consisting of N-hydroxysuccinimide ester, sulfo-N-hydroxysuccinimide ester or a salt thereof, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl and N-hydroxybenzotriazole ester. That is, E is N-succinimidyloxycarbonyl, pnitrophenoxycarbonyl, pentafluorophenoxycarbonyl, tetrafluorophenoxycarbonyl, N-benzotriazolyloxycarbonyl, or sulfo-N-succinimidyloxycarbonyl or a sodium salt thereof.

As defined above, antigen refers to any substance that can be bound in a way that is immunospecific to some degree and that can promote a humoral immune response, a cell-measured response, or both. Exemplary antigens include peptides, polypeptides, proteins, glycoproteins, lipids, glycolipids, polysaccharides, carbohydrates, polynucleotides, prions, oligonucleotides (eg, CpG), DNA, viruses, bacteria, fungi, parasites, toxins, or toxoids).

In some embodiments, including any of the above embodiments of conjugates where A, p, R ', R, or E are defined, the antigen is a protein.

In some embodiments, including any of the above embodiments of conjugates where A, p, R ', R, or E are defined, the antigen is a lipid.

<img file="MX347240B_D0018.tif" />

In some embodiments, including any of the above embodiments of conjugates where A, p, R ', R, or E are defined, the antigen is a vaccine.

In some embodiments, including any of the above embodiments of conjugates where A, p, R ', R, or the antigen is defined, the antigen is covalently bound to the linker at ** through an amide, disulfide, urea, thiourea , carbamate, or a carbon-sulfur or carbononitrogen bond in the alpha position to an amide or sulfone or directly bonded to a succinimide ring. In some embodiments, the antigen is covalently linked to the linker at ** through an amide or a carbon-sulfur or carbon-nitrogen bond in the alpha position to an amide or sulfone or directly linked to a succinimide ring. In some embodiments, the antigen is covalently linked to the linker at ** through an amide functional group.

In some embodiments, the linker is a compound represented by the formula:

<img file="MX347240B_D0019.tif" />

where A is CH or N, p is in a range from 1 to

50, and LG is a group that can be displaced by an amine. When this linker is used to modify an antigen, a modified antigen that has at least one segment

<img file="MX347240B_D0020.tif" />

represented

IMPI

Mexican INSTITUTE OF INDUSTRIAL PROPERTY by the formula can be provided:

<img file="MX347240B_D0021.tif" />

C (O) -NH- (CH₂CHjO)_p-CH2CH₂-C (O) -N * (H) where A is CH or N, p is in a range from 1 to

50, and the nitrogen atom indicated by N * is covalently bound to the antigen.

In some embodiments of the linker or modified antigen, A is CH or N. In some embodiments, A is CH.

In some modalities, including any of

<td>the</td><td colspan="2">modalities</td><td>previous</td><td colspan="4">of the linker</td><td colspan="4">or antigen</td>

<td colspan="3">modified where</td><td colspan="2">A is defined,</td><td colspan="3">p is in a</td><td>interval</td><td>of</td><td> 1</td><td>to</td>

<td> 50 .</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 2</td><td>to</td>

<td> 50 .</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 1</td><td>to</td>

<td> 40 .</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 2</td><td>to</td>

<td> 40.</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 1</td><td>to</td>

<td> 30.</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 2</td><td>to</td>

<td> 30 .</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 2</td><td>to</td>

<td> 24 .</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 2</td><td>to</td>

<td> 16 .</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 2</td><td>to</td>

<td> 12 .</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 4</td><td>to</td>

<td> 24 .</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 4</td><td>to</td>

<td> 16 . 12 .</td><td>In</td><td>some</td><td>modalities,</td><td>P</td><td>this</td><td>in</td><td>a</td><td>interval</td><td>of</td><td> 4</td><td>to</td>

In some modalities, including any of the previous modalities of the linker where A or op is

<img file="MX347240B_D0022.tif" />

IMPI

MEXICAN INSTITUTE

OF INDUSTRIAL CURRENCY defined,

LG is a group that can be displaced by an amine. In some embodiments, LG is selected from the group consisting of N-succinimidyloxy, p-nitrophenoxy, pentafluorophenoxy, tetrafluorophenoxy, N-benzotriazolyloxy, and sulfo-N-succinimidyloxy or a sodium salt thereof. In some embodiments, LG is -Cl, -Br, or -I.

In the modified antigen, the antigen can be any of those previously described. In some embodiments, including any of the above embodiments of the modified antigen where A or p is defined, the antigen is a protein.

In some embodiments, including any of the above embodiments of conjugates where A or p is defined, the antigen is a lipid.

In some embodiments, including any of the above conjugates where A or p is defined, the antigen is a vaccine.

Any suitable IRM compound may be useful in providing the conjugates of the present invention. Suitable MRI compounds include small organic molecules, that is, molecules having a molecular weight of less than about 1000 Daltons, although in some embodiments a suitable MRI compound may have a molecular weight of less than about 700 Daltons. In some embodiments, a suitable MRI compound may have a weight⁸ IMPIO ^

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL molecular weight from about 500 Daltons to about 700 Daltons, whereas in other embodiments, a suitable MRI compound may have a molecular weight from about 250 to about 500 Daltons.

Suitable MRIs include the compounds described in, for example, US Patent Nos. 4,689,338; 4,929,624; 5,266,575; 5,268,376; 5,346,905; 5,352,784; 5,389,640; 5,446,153; 5,482,936; 5,756,747;

6,110,929; 6,194,425; 6,331,539; 6,376,669; 6,451,810;

6,525,064; 6,541,485; 6,545,016; 6,545,017; 6,573,273;

6,656,938; 6,660,735; 6,660,747; 6,664,260; 6,664,264;

6,664,265; 6,667,312; 6,670,372; 6,677,347; 6,677,348;

6,677,349; 6,683,088; 6,756,382; 6,797,718; and 6,818,650; United States Patent Publications Nos.

2004/0091491; 2004/0147543; 2004/0176367; and 2006/0100229; and International Publications Nos. WO2005 / 18551, WO2005 / 18556, WO2005 / 20999, WO2005 / 032484, WO2005 / 048933, WO2005 / 048945, WO2005 / 051317, WO2005 / 051324, WO2005 / 066169, WO2005 / 066170, WO2005 / 066172, WO2005 / 0783, WO2005 / 0783 079195, WO2005 / 094531, WO2005 / 123079, WO2005 / 123080, WO2006 / 009826, WO2006 / 009832, WO2006 / 026760, WO2006 / 028545, WO2006 / 028962, WO2006 / 0291 15, WO2006 / 038923, WO2003 / 065280 , WO2006 / 083440, WO2006 / 086449, WO2006 / 086633, WO2006 / 086634, WO2006 / 091394, WO2006 / 091567, WO2006 / 091568, WO2006 / 091647, WO2006 / 093514, WO2006 / 098852, WO2006 / 107771, WO2006 / 107851, and

WO2006 / 107853.

IMPI

MEXICAN INSTITUTE

DE La fropudal · INDUSTRIAL

<img file="MX347240B_D0023.tif" />

Additional examples of ¿Tea lILUléLuler - * - ^ * -³ Suitable small derivatives include certain purine derivatives (such as those described in US Patent Nos. 6,376,501, and 6,028,076), certain imidazoquinoline amide derivatives (such as those described in US Patent No. 6,069,149), certain imidazopyridine derivatives (such as those described in U.S. Patent No. 6,518,265), certain benzimidazole derivatives (such as those described in US Patent 6,387,938), certain derivatives of a 4-aminopyrimidine fused to a five-membered nitrogen-containing heterocyclic ring (such as the adenine derivatives described in US patents the United States Nos. 6,376,501; 6,028,076 and 6,329,381; and in WO2002 / 08905), certain 3-β-ϋribofuranosylthiazol [4,5-d] pyrimidine derivatives (such as those disclosed in US Publication No.

2003/0199461), and certain small molecule immunoenhancing compounds such as those described, for example, in US2005 / 0136065.

Other suitable MRIs include large biological molecules such as oligonucleotide sequences. Some MRI oligonucleotide sequences contain cytokine-guanine dinucleotides (CpG) and are described, for example, in

ΙΜΡΙ (^&gt;

Mexican iNsrnyro 'nor LA RROMEDAD industrial the United States Patents Nos. 6,194,388; 6,207,646;

6,239,116; 6,339,068; and 6,406,705. Some CpG-containing oligonucleotides may include synthetic immunomodulatory framework moieties such as those described, for example, in US Pat. Nos.

6,426,334 and 6,476,000. Other IRM nucleotide sequences lack the CpG sequences and are described, for example, in International Patent Publication No. WO2000 / 75304. Other MRI nucleotide sequences include guanosine and uridine rich single stranded RNA (ssRNA) such as those described, for example, in Heil et al.,

Science, vol. 303, pp. 1526-1529, March 5, 2004.

Other suitable MRIs include biological molecules such as aminoalkyl glucosaminide phosphates (AGPs) and are described, for example, in US Patent Nos. 6,113,918; 6,303,347; 6,525,028; and 6,649,172.

In some embodiments of the present invention, a suitable IRM compound may be an agonist of at least one TLR such as TLR7 or TLR8. In some embodiments, MRI can also be a TLR 9 agonist.

In some embodiments of the present invention, a suitable IRM compound may include a 2-aminopyridine ring fused to a five-membered nitrogen-containing heterocyclic ring or a 4-aminopyrimidine ring fused to a heterocyclic ring that "Trryro Mexicano Di LA PXOniDAD industrial vv¹»Contains nitrogen, five-membered. ~

Suitable IRM compounds include "Toscompounds containing a 2-aminopyridine ring fused to a five-membered nitrogen-containing heterocyclic ring. Such compounds include the imidazoquinoline amines, for example the substituted imidazoquinoline amines such as the amide substituted imidazoquinoline amines, the sulfonamide substituted imidazoquinoline amines, the urea substituted imidazoquinoline amines, the aryl ether substituted imidazoquinoline amines, the imidazoquinoline-amines substituted with heterocyclic ether, imidazoquinoline-amines substituted with amido ether, sulfonamido ether-substituted imidazoquinoline-amines, urea-substituted imidazoquinoline-ethers, thioether-substituted imidazoquinoline-amines, hydroxylamine-substituted imidazoquinoline-amines, oxime-substituted imidazoquinoline-amines, 6-substituted imidazoquinoline-amines 7-, 8-, or 9-aryl, heteroaryl, aryloxy or arylalkyleneoxy, imidazoquinolinamines and imidazoquinoline diamines; tetrahydroimidazoquinoline amines such as amide substituted tetrahydroimidazoquinoline amines, sulfonamide substituted tetrahydroimidazoquinoline amines, urea substituted tetrahydroimidazoquinoline amines, ether substituted tetrahydroimidazoquinoline amines

<img file="MX347240B_D0024.tif" />

aryl, heterocyclic ether-substituted tetrahydroimidazoquinoline-amines, amide-ether-substituted tetrahydroimidazoquinoline-amines, sulfonamido-ether-substituted tetrahydroimidazoquinolinamines, urea-substituted tetrahydroimidazoquinoline-amines, amide-ether-substituted tetrahydroimidazoquinro-ethers with hydroxylamine, oxime-substituted tetrahydroimidazoquinoline-amines, and the tetrahydroimidazoquinolineindiamines; imidazopyridine amines such as amide substituted imidazopyridinamines, sulfonamide substituted imidazopyridine amines, urea substituted imidazopyridine amines, aryl ether substituted imidazopyridine amines, imidazopyridine substituted amines with heterocyclic ether substituted imidazopyridine amines, amide ether, sulfonamido ether substituted imidazopyridine amines, urea substituted imidazopyridine ethers, and thioether-substituted imidazopyridine amines;

1,2-bridged imidazoquinoline amines;

6,7-fused cycloalkylimidazopyridine amines;

imidazonaphthyridine amines; tetrahydroimidazonaphthyridine amines; oxazoloquinoline amines; thiazoloquinoline amines;

oxazolopyridine amines; thiazolopyridine amines;

oxazolonaphthyridine amines; pyrazolopyridine amines;

thiazolonaphthyridine amines;

pyrazoloquinoline amines;

IMPI ^ ntrrnvro Mexican

OF THE INDUSTRIAL PROPERTY * ° · »^ tetrahydropyrazoloquinoline-amines; pyrazolonaphthyridine amines;

tetrahydropyrazolonaphthyridine amines; and 1H-imidazo dimers fused to pyridine amines, guignolin amines, tetrahydroquinoline amines, naphthyridine amines, or tetrahydronaphthyridine amines.

In some embodiments, the MRI compound is an imidazonaphthyridine amine, a tetrahydroimidazonaphthyridineamine, an oxazoloquinoline amine, a thiazoloquinoline amine, an oxazolopyridine amine, a thiazolopyridine amine, an oxazolonaphthyridine amonazoline, an amonazolonaphthyridine amoneidine amine , a pyrazoloquinoline amine, a tetrahydropyrazoloquinoline amine, a pyrazolonaphthyridinamine, or a tetrahydropyrazolonaphthyridine amine.

In some embodiments, the IRM compound is a substituted imidazoquinoline amine, a tetrahydroimidazoquinoline amine, an imidazopyridine amine, a 1,2-bridged imidazoquinoline amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazonaphthyridine amine, a tetrahydroimidazonaphthyridinamine, an oxazoloquinoline amine, a thiazoloquinoline amine, an oxazolopyridine amine, a thiazolopyridine amine, an oxazolonaphthyridine amine, a thiazolonaphthyridine amine, a pyrazolopyridine amine, a pyrazoloquinolin amine, a tetrahydropyrazoloquinoline amine, a pyrazolonaphthyridine amine, or a tetrahydropyrazolonaphthyridine amine.

In some embodiments, imidazoquinoline amine, a

<img file="MX347240B_D0025.tif" />

imidazonaphthyridine amine, pyrazoloquinoline amine, a pyrazolonaphthyridine amine, or a thiazoloquinoline amine.

<td>How I know</td><td>used in</td><td>the present,</td><td>a</td>

<td>imidazoquinolin-amine</td><td>replaced</td><td>refers to</td><td>a</td>

<td>imidazoquinolin-amine</td><td>replaced</td><td>with amide,</td><td>a</td>

<td>imidazoquinolin-amine</td><td>replaced</td><td>with sulfonamide,</td><td>a</td>

<td>imidazoquinolin-amine</td><td>replaced</td><td>with urea,</td><td>a</td>

<td>imidazoquinolin-amine</td><td colspan="2">substituted with aryl ether,</td><td>a</td>

<td>imidazoquinolin-amine</td><td>replaced with</td><td>heterocyclic ether,</td><td>a</td>

<td>imidazoquinolin-amine</td><td colspan="2">substituted with amido ether,</td><td>a</td>

<td>imidazoquinolin-amine</td><td colspan="3">substituted with sulfonamido ether,</td>

<td>imidazoquinoline-ether</td><td>replaced</td><td>with urea,</td><td>a</td>

<td>imidazoquinolin-amine</td><td>replaced</td><td>with thioether,</td><td>a</td>

<td>imidazoquinolin-amine</td><td colspan="2">substituted with hydroxylamine,</td><td>a</td>

<td>imidazoquinolin-amine</td><td>replaced</td><td>with oxime,</td><td>a</td>

<td>imidazoquinolin-amine</td><td>replaced with</td><td colspan="2">6-, 7-, 8-, or 9-aryl,</td>

heteroaryl, aryloxy, or arylalkyleneoxy, or an imidazoquinoline diamine. In some embodiments, the substituted imidazoquinoline amines exclude l- (2-methylpropyl) -lH-imidazo [4,5-c] quinolin-4-amine and 4-amino-α, adimethyl-2-ethoximathyl-lH-imidazo [ 4,5-c] quinolin-l-ethanol.

Unless otherwise indicated, reference to a compound may include the compound in any form.

<img file="MX347240B_D0026.tif" />

including any isomer

IMPI Mexican institute DE LA PROPIEDAD INDUSTRIAL pharmaceutically acceptable, (for example, diastereomer or enantiomer), salt, solvate, polymorph, and the like. In particular, if a compound is optically active, reference to the compound can include each of the enantiomers of the compound, as well as racemic mixtures of the enantiomers.

IRM compounds, including any of the specific IRM compounds described above, include a hydrazine or hydrazide substituent. The hydrazine or hydrazide substituent can be linked to the IRM compound (eg, in some embodiments, an imidazoquinolinamine, imidazonaphthyridine-amine, imidazopyridine-amine, pyrazoloquinoline-amine, pyrazolonaphthyridine-amine, or pyrazolopyridine-amine) at position 1. In some of these modalities, the MRI is formula I or II:

<img file="MX347240B_D0027.tif" />

I Π where

R_to and R_b are each independently selected from the group consisting of:

hydrogen, halogen,

IMPI

INSTITUTO MEMCANO DE LA PROPIEDAD INDUSTRIAL alkyl alkenyl, alkoxy, alkylthio, and

-N (R₉)₂;

or when taken together, R_TO and R_B form a fused heteroaryl ring containing a heteroatom selected from the group consisting of nitrogen and sulfur, or a fused aryl ring wherein the aryl or heteroaryl ring is unsubstituted or substituted with one more R, or substituted with one R group₃, or substituted with a group R₃ and a group R;

or when taken together, R_TO and R_B form a 5 to 7 membered saturated fused ring, optionally containing a heteroatom selected from the group consisting of nitrogen and sulfur, and unsubstituted or substituted with one or more R groups;

R is selected from the group consisting of: halogen, hydroxyl, alkyl, alkenyl, haloalkyl, alkoxy, alkylthio, and ²⁷ IMPI ^ Mexican institute

OF INDUSTRIAL PROPERTY

-N (R₉) ₂; __________________

R₂ is selected from the group consisting of: amino, -R₄, -xr₄, -XYR_4z and

-XR₅;

R₃ is selected from the group consisting of:

-ZR₄,

-zxr₄,

-zxyr₄,

-ZXYXYR₄, and

-ZXR₅',

X is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, heteroarylene, and heterocyclylene wherein the alkylene, alkenylene, and alkynylene groups may be optionally interrupted or terminated by arylene, heteroarylene, or heterocyclylene, interrupted by one or more groups -O-, or terminated by -O or -N (H) -;

And it is selected from the group consisting of:

-OR-,

-SW)₀-2-,

-SW)₂-N (R₈) -,

-C (R₆) -,

<img file="MX347240B_D0028.tif" />

IMPI

MEXICAN INSTITUTE

Dt THE INDUSTRIAL RAMBITY

-C (R₆) -O-,

-oc (r₆)

-OC (O) -o-,

-N (Re) -Q-,

-c (r₆) -n (r₈)

-OC (R₆) -n (r₈)

-C (R₆) -n (or₉)

-ON (R₈) -Q-,

-ON = C (R₄) - ,

-C (= NOR₈)

<img file="MX347240B_D0029.tif" />

<img file="MX347240B_D0030.tif" />

X 'is selected from the group consisting of -X-, -XC (O) -, -XYX-, and -XYXC (O)

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX347240B_D0031.tif" />

Z is a bond or -0-;

R₄ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkylenyl, aryloxyalkylenyl, alkylarylenyl, heteroaryl, heteroarylalkylenyl, heteroaryloxyalkylenyl, alkylheteroarylenyl, and heterocyclyl in which the alkyl, alkylalkenyl, arylenyl, arylalkyl, alkynyl, arylenyl, groups , heteroaryl, heteroarylalkylenyl, heteroaryloxyalkylenyl, alkylheteroarylenyl, and heterocyclyl may be unsubstituted or substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, hydroxyalkyl, haloalkyl, haloalkoxy, halogen, nitro, hydroxy, mercapto, cyano, aryl, aryloxy, arylalkyleneoxy, heteroaryl, heteroaryloxy, heteroarylalkyleneoxy, heterocyclyl, amino, alkylamino, dialkylamino, (dialkylamino) alkyleneoxy, and in the case of alkyl, alkenyl, alkynyl, and heterocyclyl, oxo;

R₅ is selected from the group consisting of

<img file="MX347240B_D0032.tif" />

R₆ is selected from the group consisting of = O and = S;

IMPI ^

MEXICAN INSTITUTE

DS INDUSTRIAL PIOPIGITY

R₇ is alkylene of 2 to 7 carbon atoms;

R₈ is selected from the group consisting of hydrogen, alkyl, alkoxyalkylenyl, hydroxyalkylenyl, arylalkylenyl, and heteroarylalkylenyl;

R₉ is selected from the group consisting of hydrogen and alkyl;

Rio is alkylene of 3 to 8 carbon atoms;

A is selected from the group consisting of -O-, C (O) -, -S (0) or-2-, and -N (R₄) -;

A 'is selected from the group consisting of -0-, S (O)_or_₂-, -N (-QR₄) -, and -CH₂-; Q is selected from the group consisting of a bond, -C (R₆) -, -C (R₆) -C (R₆) -, -S (O)₂-, -C (R₆) N (R₈) -W-, -S (O)₂-N (R₈) -, -C (R₆) -O-, -C (R₆) -S-, and -C (R₆) N (0R₉) - ;

V is selected from the group consisting of -C (R₆) -, -OC (R₆) -, -N (R₈) -C (R₆) -, and -S (0)₂-;

W is selected from the group consisting of a bond, -C (0) -, and -S (0)₂-; y a and b are independently integers from 1 to 6, provided that a + b is &lt;7.

As used herein, the terms alkyl, alkenyl, alkynyl, and the prefix ale- are inclusive of straight chain and branched chain groups and cyclic groups, eg, cycloalkyl and cycloalkenyl. Unless specified

IMPI

MEXICAN INSTITUTE

FROM INDUSTRIAL PROPERTY otherwise, these groups contain 1 to 2 0 carbon atoms, with the alkenyl groups containing 2 to 20 carbon atoms, and the alkynyl groups containing 2 to 20 carbon atoms. In some embodiments, these groups have up to a total of 10 carbon atoms, up to 8 carbon atoms, up to 7 carbon atoms, up to 6 carbon atoms, or up to 4 carbon atoms. Cyclic groups can be monocyclic or polycyclic and preferably have 3 to 10 carbon atoms in the ring. Exemplary cyclic groups include substituted and unsubstituted cyclopropyl, cyclopropylmethyl, cyclopentyl, cyclohexyl, adamantyl, and bornyl, norbornyl, and norbornenyl.

Unless otherwise specified, alkylene, alkenylene, and alkynylene are the divalent forms of the alkyl, alkenyl, and alkynyl groups defined above. The terms, alkylenyl, alkenylenyl, and alkynylenyl are used when the alkylene, alkenylene, and alkynylene groups, respectively, are substituted. For example, an arylalkylenyl group comprises an alkylene moiety to which an aryl group is attached.

The term "haloalkyl" is inclusive of groups that are substituted with one or more halogen atoms, including perflucid groups. This is also true of other groups that include the prefix³² IMPI

MEXICAN INSTITUTE

OF THE PROPERTY

INDUSTRIAL - halo-. Examples of haloalkyl groups · βιί «ιαΗΐ1θ? they include chloromethyl and trifluoromethyl.

An alkylene group with carbon atoms optionally interrupted by -O- refers to having carbon atoms on either side of -O-. An example is -CH₂-CH₂-O-CH₂-CH₂-.

An alkylene group with carbon atoms optionally terminated by -0- refers to having the -O- on either end of the alkylene group or the chain of carbon atoms. Examples include -O-CH₂-CH₂-CH₂CH₂- and -CH₂-CH₂-CH₂-CH₂-O-. In the compounds of Formulas I and II and the conjugates of the present invention, when X 'is -O- terminated alkylene, the -0- may be connected either to the nitrogen of the imidazole ring or to the group Y. In the compounds of Formulas I and II and the conjugates of the present invention, when X 'is N (H) - terminated alkylene, the -N (H) - is typically connected to the imidazole ring.

The term aryl as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl, and indenyl.

Unless indicated otherwise, the term "heteroatom" refers to oxygen, sulfur, or nitrogen atoms.

The term heteroaryl

IMPI

INSTITUTO MEXICANO DE LA PRenEDAD, _ INDUSTRIAL includes the

<img file="MX347240B_D0033.tif" />

aromatic rings or ring systems that contain at least one heteroatom in the ring (eg, oxygen, sulfur, nitrogen). In some embodiments, the term heteroaryl includes a ring or ring system that contains 2-12 carbon atoms, 1-3 rings, 1-4 heteroatoms, and oxygen, sulfur, and nitrogen as the heteroatoms. Exemplary heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrimidalinyl, benzoxazolyl, pyrimidinyl, benzoxazolyl, pyrimidinyl. , isoxazolyl, isothiazolyl, purinyl, quinazolinyl, pyrazinyl, 1oxydopyridyl, pyridazinyl, triazinyl, tetrazinyl, oxadiazolyl, thiadiazolyl, and so on.

The term "heterocyclyl" includes non-aromatic rings or ring systems that contain at least one ring heteroatom (eg, oxygen, sulfur, nitrogen) and includes all fully saturated and partially unsaturated derivatives of the aforementioned heteroaryl groups. In some embodiments, the term heterocyclyl includes a ring or ring system that contains 2-12 carbon atoms, 1-3 rings, 1-4 heteroatoms, and oxygen, sulfur, and nitrogen as the heteroatoms. Exemplary heterocyclyl groups

<img file="MX347240B_D0034.tif" />

include pyrrolidinyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, 1,1-dioxothiomorpholinyl, piperidinyl, piperazinyl, thiazolidinyl, imidazolidinyl, isothiazolidinyl, tetrahydropyranyl, quinuclidinyl, homopiperidinyl (azepanzelanyl), 1,4-diopanzepanyl, homozepelanzepanyl, dioxopelanyl), , aziridinyl, azetidinyl, dihydroisoquinolin- (1H) -yl, octahydroisoquinolin- (1H) -yl, dihydroquinolin- (2H) -yl, octahydroquinolin- (2H) -yl, dihydro-1H-imidazolyl, 3azabicyclo [3.2.2] non -3-ilo, and the like. The term heterocyclyl includes the bicyclic and tricyclic heterocyclic ring systems. Such ring systems include fused and / or bridged rings and spiro rings. Fused rings can include, in addition to a saturated or partially saturated ring, an aromatic ring, for example a benzene ring. Spiro rings include two rings joined by one spiro atom and three rings joined by two spiro atoms.

When the heterocyclyl contains a nitrogen atom, the attachment point of the heterocyclyl group can be the nitrogen atom.

The terms arylene, heteroarylene, and heterocyclylene are the divalent forms of the defined aryl, heteroaryl, and heterocyclyl groups.

<img file="MX347240B_D0035.tif" />

previously. The terms arylenyl, heteroarylenyl, and heterocyclylenyl are used when arylene, heteroarylene, and heterocyclylene, respectively, are substituted. For example, an alkylarylenyl group comprises an arylene portion to which an alkyl group is attached.

When a group (or substituent or variable) is present more than once in any Formula described herein, each group (or substituent or variable) is independently selected, whether explicitly stated or not. For example, for the formula -N (R₈) -C (O) N (R₈) - each R group₈ is independently selected. In yet another example, when two groups R₁₀ are present, each R group₁₀ is independently selected.

In some modalities of Formulas I and II, when taken together, R_TO and R_B they form a fused aryl ring that is unsubstituted. In some of these embodiments, the fused aryl ring is a fused benzene ring.

In some modalities of Formulas I and II, including the modalities where R_TO and R_b are as defined above, R₂ is hydrogen, amino, alkyl, alkoxyalkylenyl, alkylaminoalkylenyl, or hydroxyalkylenyl.

In some modalities of Formulas I and II, including the modalities where R_TO and R_B they are as defined

<img file="MX347240B_D0036.tif" />

IMPI

INSTITUTO MEXICANO D »LA RROMtDAD INDUSTRIAL previously, R₂ is hydrogen, alkyl, alkoxyalkylenyl, or hydroxyalkylenyl.

In some modalities of Formulas I and II, including the modalities where R_TO and R_B as defined above, R₂ it is hydrogen, alkyl, or alkoxyalkylenyl.

In some modalities of Formulas I and II, including the modalities where R_TO and R_B and R₂ as defined above, X 'is -X'-YX₂- or -X_x-YX₂-C (O) -, where X_xis alkylene optionally interrupted with one or more -0- groups and optionally terminated by -0-; Y is -NH-C (O) -, and X₂it is alkylene, arylene, or heteroarylene.

In some modalities of Formulas I and II, including the modalities where R_TO and R_B and R₂ as defined above, X 'is -X ^ YX²-, where X¹ is alkylene optionally interrupted with one or more -0- groups and optionally terminated by -O-; Y is -NH-C (O) -, and X² it is phenylene or pyridylene.

In some embodiments, compounds of Formula I are described in co-pending United States Patent Application Serial No. 61 / 493,051, filed June 3, 2011, and incorporated by reference herein in its entirety.

In some embodiments, the compound of Formula I is N- (4- {[4-amino-2-butyl-1H-imidazo [4,5-c] quinolin-1-

<img file="MX347240B_D0037.tif" />

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula

I is N- (4 - {[4-amino-2-butyl-lH-imidazo [4,5-c] quinolin-1-yl] oxy} butyl) -6- (Ν'-isopropylidenehydrazino) nicotinamide:

<img file="MX347240B_D0038.tif" />

or a pharmaceutically acceptable salt thereof.

<img file="MX347240B_D0039.tif" />

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL HUMIDITY

In some embodiments, the compound of Formula

I is N- {2- [4-Amino-2- (ethoxymethyl) -IH-imidazo [4,5-c] quinolin-1-yl] -1,1-dimethylethyl} -4-hydrazino-4-oxobutanamide:

<img file="MX347240B_D0040.tif" />

or a pharmaceutically salt

In some modalities, the acceptable of the same.

Formula compound

I is N- (4- {[4-amino-2-butyl-lH-imidazo [4,5-c] quinolin-1-yl] oxy} butyl) -4- (Ν'-isopropylidenehydrazino) benzamide:

<img file="MX347240B_D0041.tif" />

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula

I is N- (4- {[4-amino-2-butyl-1H-imidazo [4,5-c] quinolin-139

<img file="MX347240B_D0042.tif" />

IMPI

MEXICAN INSTITUTE

DE LA MOHEDAL · vdustsial il] oxy} butyl) -4-hydrazinobenzamide:

<img file="MX347240B_D0043.tif" />

or a pharmaceutically acceptable salt thereof.

The hydrazine or hydrazide substituent can be linked to the IRM compound (eg, in some embodiments, an imidazoquinoline amine, imidazonaphthyridine amine, pyrazoloquinoline amine, pyrazolonaphthyridine amine, or thiazoloquinoline amine) at position 7 or at position 8.

In some modalities, the MRI is formula III, IV, or V:

<img file="MX347240B_D0044.tif" />

where R₂, X ', and Z are as defined

<img file="MX347240B_D0045.tif" />

ΙΝΓΠΤυΤΟ MEXICANO Di LA PROPERTY INDUSTRIAL above, A is CH or N, and Ri is selected from the group consisting of:

-N (H) -R₄,

-or₄,

-r₄,

-xr₄,

-xyr₄,

-N (H) -XYR₄,

-XYXYR₄, and

-XR₅;

where X, Y, R₄, and R₅ they are as defined above.

In some embodiments of Formulas III, IV, and V, the group -Z-X'-NHNH₂ is linked at position 7. In some embodiments of Formulas III, IV, and V, the group Z-X'-NHNH₂ is linked at position 8.

In some modalities of Formulas III, IV, and V, including any of the previous modalities, A is CH. In other modes, A is N.

In some modalities of Formulas III, IV, and V, including the modalities where A is defined as described above, R₂ is hydrogen, amino, alkyl, alkoxyalkylenyl, alkylaminoalkylenyl, or hydroxyalkylenyl.

In some modalities of Formulas III, IV, and

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

V, including modalities where A is defined as described above, R₂ is hydrogen, alkyl, alkoxyalkylenyl, or hydroxyalkylenyl.

In some modalities of Formulas III, IV, and V, including the modalities where A is defined as described above, R₂ it is hydrogen, alkyl, or alkoxyalkylenyl.

In some modalities of Formulas III, IV, and V, including the modalities where A and R₂ as defined above, Z is -0-. In some embodiments, Z is a link.

In some modalities of Formulas III, IV, and

V, including modalities where A, Z, and R₂ as defined above, X 'is -X¹-YX²- or -X¹-YX²-C (O) -, where X¹ is alkylene optionally interrupted with one or more -0- groups and optionally terminated by -0-; Y is -NHC (0) -, and X² it is alkylene, arylene, or heteroarylene.

In some modalities of Formulas III, IV, and

V, including modalities where A, Z, and R₂ as defined above, X 'is -X¹-YX²-, where X¹ is alkylene optionally interrupted with one or more groups -0 and optionally terminated by -0-; Y is -NH-C (O) -, and X² it is phenylene or pyridylene.

In some modalities of Formulas III, IV, and

V, including modalities where A, Z, X ', and R₂ how I know

<img file="MX347240B_D0046.tif" />

IMPI

INSTrfU * O¡J ** KANO I heard the iNtíuynuxL defined above, R_x is selected from the group consisting of alkyl, arylalkylenyl, aryloxyalkylenyl, hydroxyalkyl, dihydroxyalkyl, alkylsulfonylalkylenyl, -XYR₄, -XR₅, and heterocyclylalkylenyl, wherein the heterocyclyl of the heterocyclylalkylenyl group is optionally substituted with one or more alkyl groups; where X is alkylene; Y is -N (R₈) -C (0) -, -N (R₈) -S (O) ₂-, N (Rs) -C (O) -N (R₈)or

<img file="MX347240B_D0047.tif" />

R₄ is alkyl, aryl, or heteroaryl; and R₅ is

<img file="MX347240B_D0048.tif" />

In some modalities of Formulas III, IV, and

V, including modalities where A, Z, X ', and R₂ as defined above, R_x is selected from the group consisting of 2-hydroxy-2-methylpropyl, 2-methylpropyl, propyl, ethyl, methyl, 2,3-dihydroxypropyl, 2-phenoxyethyl, 4 - [(methylsulfonyl) amino] butyl, 2-methyl-2 [(methylsulfonyl) amino] propyl, 2- (acetylamino) -2methylpropyl, 2- {[(isopropylamino) carbonyl] amino} -2 methylpropyl, 4 - {[(isopropylamino) carbonyl] amino} butyl, 4 (1, l- Dioxydoisothiazolidin-2-yl) butyl, tetrahydro-2H-pyran-4ylmethyl, and (2,2-dimethyl-1,3-dioxolan-4-yl) methyl.

IMPIOUS*

MEXICAN INSTITUTE OF CURRENCY OR INDUSTRIAL

Preparation of Conjugates

IRM compounds and linkers useful in practicing the present invention can be synthesized by synthetic routes that include processes analogous to those well known in the chemical arts, particularly in light of the disclosure contained herein. Starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, Wisconsin, USA) or are readily prepared using methods well known to those skilled in the art (for example, prepared by methods generally described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley, New York, (1967-1999 ed.); Alan R. Katritsky, Otto Meth-Cohn, Charles W. Rees, Comprehensive Organic Functional Group Transformations, v 1-6, Pergamon Press, Oxford, England, (1995); Barry M. Trost and Ian Fleming, Comprehensive Organic Synthesis, v. 1-8, Pergamon Press, Oxford, England, (1991); or Beilsteins Handbuch der organischen Chemie, 4, Aufl. Ed. Springer-Verlag, Berlin, Germany, including supplements (also available via the Beilstein online database).

For illustrative purposes, the reaction schemes described below provide potential routes to synthesize IRM compounds and useful linkers to

<img file="MX347240B_D0049.tif" />

<img file="MX347240B_D0050.tif" />

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY . practice the present invention as well as key intermediaries. For a more detailed description of the individual reaction steps, see the EXAMPLES section below. Those skilled in the art will appreciate that other synthetic routes can be used to synthesize the IRM compounds and linkers. Although starting materials and specific reagents are described in the reaction schemes and discussed below, other starting materials and reagents can be readily substituted to provide a variety of derivatives and / or reaction conditions. Furthermore, many of the compounds prepared by the methods described below can be further modified in light of this disclosure, using conventional methods well known to those skilled in the art.

In preparing IRM compounds and linkers useful in practicing the present invention it may sometimes be necessary to protect a particular functional group while other functional groups are reacted on an intermediate. The need for such protection will vary depending on the nature of the particular functional group and the conditions of the reaction step. Suitable amino protecting groups include acetyl, trifluoroacetyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl, and 9-fluorenylmethoxycarbonyl (Fmoc). The

IMPI ^

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OF PROPERTY C * »·?

INDUSTRIAL ^ C ^ L. Suitable hydroxyl protecting groups include acetyl and silyl groups such as the tert-butyl-dimethylsilyl group. For a general description of protecting groups and their use, see TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, John Wiley &amp; Sons, New York, United States, 1991.

Conventional separation and purification methods and techniques can be used to isolate IRM compounds and linkers, as well as various intermediates related to them. Such techniques can include, for example, all types of chromatography (high performance liquid chromatography (HPLC), column chromatography using common absorbents such as silica gel, and thin-layer chromatography), recrystallization and differential extraction techniques (i.e. say, liquid-liquid).

In Reaction Scheme I, intermediate compounds useful for preparing the hydrazine or hydrazide substituted immune response modifiers are described. In step (1) of Reaction Scheme I, the hydrazinobenzoic acid or hydrazinonicotinic acid compound of Formula VIII is reacted with acetone at room temperature to provide the hydrazone-substituted compound of Formula IX. The initial hydrazine substituted compounds of Formula VIII are

<img file="MX347240B_D0051.tif" />

<img file="MX347240B_D0052.tif" />

INSTITUTO MEXICANO „Ol LA 4-hydrazmobenzoic acid (VIII where A = CH) and hydrazinonicotinic acid (VIII where A = N). These · '' CUItipUédLos can be prepared using the reaction conditions described by Lagisetty, P.; Vilekar, P.; and Awasthi, V. Biorganic and Medicinal Chemistry Letters, 19, pp. 4764-4767 (2009) or Pegurier, C; Collart, P.; Danhaive, P .; Defays, S .; Gillard, M.; Gilson, F .; Kogej, T .; Pasau, P.; Van Houtvin, N.; Van Thuyne, M.; Van Keulen, B. Bioorganic and Medicinal Chemistry Letters, 17, pp. 4228-4231 (2007); or reported in WO2006071940 (Flynn et al.).

In step (2) of Reaction Scheme I, the compound of Formula IX is reacted at room temperature with an N-hydroxysuccinimide and a standard coupling reagent such as 1,3-dicyclohexylcarbodiimide (DCC) or 1- [3 - (dimethylamino) propyl] -3-ethylcarbodiimide (EDC) in a suitable solvent such as dichloromethane or pyridine. The product of Formula X can be isolated using conventional means.

In step (3) of Reaction Scheme I, the hydrazinobenzoic acid or hydrazinonicotinic acid compound of Formula XI is reacted with acetone at room temperature to provide the hydrazone-substituted compound of Formula XII. The hydrazine-substituted compounds of Formula XI are 3-hydrazinobenzoic acid (XI where A = CH) and 5-hydrazinonicotinic acid (XI where A =

<img file="MX347240B_D0053.tif" />

These compounds can be prepared according to

Ν).

procedures in

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INSTITUTO MEXICANO DE LA FMOPIEDaO INDUSTRIAL references provided to prepare the compounds of Formula VIII.

In step (4) of Reaction Scheme I, the compound of Formula XII is reacted at room temperature with N-hydroxysuccinimide using, for example, the conditions previously described for step (2).

Reaction Scheme I

<img file="MX347240B_D0054.tif" />

In some embodiments, IRM compounds useful to make the conjugates of the present invention can be prepared according to Reaction Scheme II, where R_TO, R_B, and R2 are as defined above, X¹ is alkylene optionally interrupted with one or more -O- groups and optionally terminated by -O; and A is CH or N.

<img file="MX347240B_D0055.tif" />

In step (1) of Reaction Scheme II, a compound of Formula XIV is reacted with a compound of Formula IX (from Reaction Scheme I where A = CH or N) to provide a compound of Formula XV . The reaction can be conducted at room temperature in a solvent such as dichloromethane, pyridine, or 1-butanol with a standard coupling reagent such as 1,3-dicyclohexylcarbodiimide (DCC) or l- [3 (dimethylamino) propyl] 3-ethylcarbodiimide (EDC). ). A compound of Formula XV can be isolated using conventional methods. As an alternative method for step (1) of Reaction Scheme II, a compound of Formula XIV is reacted with a compound of Formula X (from Reaction Scheme I where A = CH or N) to provide a compound of Formula XV. The compound of Formula XIV can be dissolved in a suitable alcoholic solvent such as 1-butanol and the compound of Formula X can be slowly added at room temperature.

Various compounds of Formula XIV are known and / or methods for their preparation have been described; see for example, US Patent Nos. 7,648,997 (Kshirsagar, et al.); 6,660,747 (Crooks, et al.); 6,069,149 (Nanba); 7,579,359 (Krepski et al.);

<img file="MX347240B_D0056.tif" />

^4B IMPI

MEXICAN INSTITUTE

FROM AGE

INDUSTRIAL

7,163,947 (Griesgraber et al.), And International Patent Application Publication No. WO 2006/029115 (Kshirsagar et al.).

In step (2) of Reaction Scheme II, the acetamine protecting group is removed under acidic conditions to provide the compound of Formula XVI, which is a subgenus of Formula I. The reaction can be conducted in hydrochloric acid at temperature ambient or elevated (for example, 60 ° C). A product of Formula XVI can be isolated, for example, as a hydrochloride salt by lyophilization.

In step (la) of Reaction Scheme II, a compound of Formula XIV is reacted with a compound of either Formula XII or Formula XIII (from Reaction Scheme I where A = CH or N) according to Corresponding procedure described in step (1) to provide a compound of Formula XVII.

In step (2a) of Reaction Scheme II, the acetamine protecting group is removed under acidic conditions to provide the compound of Formula XVIII, which is a subgenus of Formula I. The reaction can be conducted according to the procedure described. in step (2).

<img file="MX347240B_D0057.tif" />

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INSTITUTO MEXICANO DS LA PROPERTY INDUSTRIAL

Reaction Scheme II

<img file="MX347240B_D0058.tif" />

In some embodiments the IRM compounds useful to make the conjugates of the present invention can be prepared according to the method of Reaction Scheme III where R₂, Ra, R_b, and Xi are as defined above.

In step (1) of Reaction Scheme III, a compound of Formula XIV is reacted with at least two equivalents of succinic anhydride. The reaction can be carried out in a suitable solvent such as DMF at a

<img file="MX347240B_D0059.tif" />

elevated temperature such as 100 ° C. R1 product of Formula XIX, or a pharmaceutically acceptable salt thereof, can be isolated using conventional methods.

In step (2) of Reaction Scheme III, the acid group on a compound of Formula XIX is activated with a carbodiimide reagent, for example, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), in the presence of tert-butyl carbazate. The reaction can be carried out in a suitable solvent such as dichloromethane, optionally in the presence of a base such as triethylamine or a catalyst such as N, N-dimethylpyridin-4-amine (DMAP). The protecting group on the intermediate product can be removed by treatment with an excess of an amine such as, for example, ethylenediamine, in a suitable solvent such as dichloromethane to provide the product of Formula XX. Compounds of Formula XX, or a pharmaceutically acceptable salt thereof, can be isolated using conventional methods.

In step (3) of Reaction Scheme III, the tert-butoxycarbonyl (BOC) group in a compound of Formula XX is removed under acidic conditions to provide a functionalized IRM of Formula XXI, which is a subgenus of Formula I. The reaction can be carried out by treating a solution of a compound of the

Formula XX in a suitable solvent such as dichloromethane with

IMPI ^^

MEXICAN IRSHTUTO

OF THE EAOHEDAL

INDUSTRIAL an acid such as trifluoroacetic acid at room temperature. The product of Formula XXI, or a pharmaceutically acceptable salt thereof, can be isolated using conventional methods. Some compounds of the

Formula XXI are known, for example, N- {2- [4-amino-2 (ethoxymethyl) -lH-imidazo [4,5-] guignolin-l-yl] -1,1-dimethylethyl} 4-hydrazino-4 -oxobutanamide is IRM 5 in United States Patent Application Publication No. 2009/0035323 (Stoermer et al.).

Reaction Scheme III

<img file="MX347240B_D0060.tif" />

In some embodiments, IRM compounds useful in preparing the conjugates of the present invention may ⁵³ IMPI ^^

MU1CANO INSTITUTE

OF THE NORWAD

INDUSTRIAL be prepared according to the methods described in Reaction Schemes II and III using the compounds of Formulas XXII, XXIII, XXIV, XXV, XXVI, or XXVII where Ri, R₂, R_to, Rb, X¹, A, and Z are as defined above, in lieu of a compound of Formula XIV. In some embodiments of Formulas XXII, XXIII, XXIV, and XXVI, the -ZX-NH groups₂ or -X-NH₂ They are linked in position 7 or position 8.

<img file="MX347240B_D0061.tif" />

<img file="MX347240B_D0062.tif" />

XXV XXVI xxvn

Many compounds of Formulas XXII, XXIII, XXIV, XXV, XXVI, and XXVII are known; others can be prepared using known synthetic methods. See for example, United States Patent Application Publication No. 2004/0147543; International Publication Nos. WO2005 / 020999, WO2005 / 032484, WO2005 / 079195, WO2006 / 009826, WO2006 / 038923, WO2006 / 093514, and WO2005 / 123079.

<img file="MX347240B_D0063.tif" />

⁵⁴ IMPI 'ΝΤΓΠΤΙΤΟ MEXICANO de LA PROMEBAL industrial

The linkers of the present invention and / or useful

1- ¹ 1,1 , _{t B} J_{U || 1} To make the modified antigens or conjugates of the present invention they can be prepared, for example, according to the method of Reaction Scheme IV, where p, LG, R ', R, and A are as defined above.

In step (1) of Reaction Scheme IV, an amino acid of Formula XXVIII is reacted with an activated ester of formylbenzoic acid or formylnicotinic acid to provide a formylbenzamide or formylnicotinamide represented by formula XXIX. The activated ester can be, for example, an N-hydroxysuccinimide ester, sulfo-N-hydroxysuccinimide ester or a salt thereof, the 4-nitrophenyl ester, the pentafluorophenyl ester, the tetrafluorophenyl ester, or the N-hydroxybenzotriazole ester. Some of these compounds (eg, Nsuccinimidyl-4-formyl benzoate) are commercially available. Others can be prepared by conventional methods. Some compounds of Formula XXVIII are commercially available (for example, the carboxy-PEG-amine compounds available from Thermo Scientific, Rockford, ILO, where R is a bond and R 'is ethylene), and others can be prepared by methods. known (eg, Riener, CK, et al. Anal. Chim. Acta, 497, pp. 101-114 (2003) where R is propoxy and R 'includes a propyl group). Formation of the amide can be carried out in a suitable solvent such as dichloromethane or

<img file="MX347240B_D0064.tif" />

IMPI'NDUSTIL chloroform, in the presence of a base such as triethylamine and optionally of catalytic DMAP. The reaction can be carried out at room temperature, and the product of Formula XXIX can be carried out by conventional methods.

In step (2) of Reaction Scheme IV, the carboxylic acid of Formula XXIX is converted in some embodiments to an activated ester to provide the linker of Formula XXX. In some embodiments, LG is the activated ester of Formula XXX selected from the group consisting of N-succinimidyloxy, p-nitrophenoxy, pentafluorophenoxy, tetrafluorophenoxy, N-benzotriazolyloxy, and sulfo-N-succinimidyloxy or a sodium salt thereof. The reaction can be carried out by reacting the compound of Formula XXIX with Ν, Ν, Ν ', N'-tetramethyl-O- (N-succinimidyl) uronium (TSTU) tetrafluoroborate in a suitable solvent or a combination of solvent such as

N, N-dimethylformamide and pyridine. The reaction can be carried out at room temperature. Alternatively, the compound of Formula XXIX can be treated with, for example, N-hydroxysuccinimide, sulfo-N-hydroxysuccinimide or a salt thereof (for example, a sodium salt), 4-nitrophenol, pentafluorophenol, tetrafluorophenol, or N-hydroxybenzotriazole in presence of a standard coupling agent such as DCC or EDC in a suitable solvent such as »ι * &lt;

<img file="MX347240B_D0065.tif" />

MEXICAN INSTITUTE OF INDUSTEIAL PROPERTY

<img file="MX347240B_D0066.tif" />

dichloromethane or pyridine. The product of Formula XXX can be isolated by conventional methods.

In some embodiments, step (2) of Reaction Scheme IV involves the conversion of the carboxylic acid of Formula XXIX to an acid chloride, acid bromide, or acid iodide to provide a linker of Formula XXX in which LG is -CI, -Br, or -I. The reaction can be carried out using conventional methods, for example, by treating the carboxylic acid with thionyl chloride, phosphorous trichloride, phosphorous pentachloride, oxalyl chloride or cyanuric chloride in a suitable solvent.

Reaction Scheme IV _{z Λ} _____ (1) NH₂-R ”- (CH₂CH₂OR)_p-R'-C (O) -OH ......................-....... »xxvm

C (O) -NH-R '' - (CH, CH₂OR)_p-R'-C (O) -OH ^TO XXIX

O $ ff C (O) -NH-R^H- (CH₂CH₂OR)_p-R^,-C (O) -LG

TO-'

XXX

In some embodiments, the linkers according to the present invention and / or useful for making the conjugates of the present invention can be prepared according to Reaction Scheme V, where p, LG, R ', R, and A are as

IMPIAS

INSTFrVTUMfXICANC

OF THE PROPERTY -ZV »JWjiflt INDUSTRIAL were defined above, E 'is bromoacetyl, chloroacetyl, iodoacetyl, or isocyanate.

In some embodiments, step (1) of Reaction Scheme V is useful for converting an acid chloride of Formula XXX where LG is -C1 to a compound of Formula XXXI, where E 'is bromoacetyl, chloroacetyl, or iodoacetyl. . The reaction can be carried out using conventional methods such as treating the acid chloride with diazomethane to provide a diazoacetyl compound, which can then be treated with hydrobromic acid or hydrochloric acid, for example, to provide the chloroacetyl or bromoacetyl group. .

In other embodiments, step (1) of Reaction Scheme V is useful for converting an acid chloride of Formula XXX where LG is -C1 to a primary amide by reaction with ammonia using conventional methods. A primary amide can then undergo Hofmann rearrangement in the presence of a bromide source such as N-bromosuccinimide to provide a compound of Formula XXXI where E 'is an isocyanate group.

In some embodiments, step (2) of Reaction Scheme V is useful for preparing a compound of Formula XXXII where E is a maleimide, and R^to it is alkylene that is interrupted by an amide group. For example, an activated carboxylic acid of Formula XXX can be treated with

<img file="MX347240B_D0067.tif" />

IMPI

INSTITUTO MEXICANO DE LA HUJNEOAD INDUSTRIAL aminopropylmaleimide or aminoethylmaleimide, which is typically commercially available in salt form, using conventional methods.

In other embodiments, step (2) of Reaction Scheme V is useful for preparing a compound of Formula XXXII where E is a chloroformate, -0C (0) -O-CH (Cl) CC1₃, OC (O) -O- (4-nitrophenyl), or succinimidyl carbonate, and R^toit is alkylene that is interrupted by an amide group. Such compounds can be prepared, for example, by treating activated carboxylic acid of Formula XXX with an amino alcohol (for example, aminoethanol or aminopropanol), and the resulting alcohol can be treated with the appropriate carbonic acid derivative to provide the linker. wanted.

Reaction Scheme V

C (0) -WR'XCH₂CH, 0)_p-R'-E 'A—' XXXI (1)

C (G) -_NH-R'4_CH2_CH ^

TO-'

XXX | (2) / -C (O) -NH-R- (CH, CH₁O) „- R^to-EA— “XXXII

Useful linkers for making conjugates of the

INSTITUTO MEXICANO present invention may also be elabo^Dra ^ 0 ^ i ^ t Modifiers of the Schemes of ρ.? ^ π_Ρίήη rv yy guo might be apparent to a person skilled in the art. Linkers useful for making the conjugates of the present invention can also be made starting with a commercially available poly (ethylene glycol) diamine instead of an amino acid of Formula XXVIII. One end of the diamine can be reacted according to the methods of step (1) of Reaction Scheme IV, above, and the other can be treated with known heterobifunctional crosslinkers such as 4- (maleimidomethyl) cyclohexane-1-carboxylate sulfo-Nsuccinimidyl, N- (gammamaleimidobutyryloxy) sulfosuccinimide ester, N-succinimidyl 3 (bromoacetamido) propionate, and 4-succinimidiyloxycarbonylmethyl-alpha- (2-pyridyldithio) toluene, which are commercially available for example, from Thermo Scientific.

Modified antigens (eg, antigens modified with any of the linkers described above in any of their modalities) can be prepared according to a variety of methods. Typically, the antigen has a reactive functional group that allows a reaction with the linker. For example, an antigen may have one or more (eg, typically multiple) terminal amino groups from the⁶⁰ IMPI ^

INSTITUTO MEXICANO DE LA WOHEDAO industrial lysine residues that can be reactive, for example, with an E group (for example, activated carboxylic acid group) on the linker. It will be appreciated by those of skill in the art that in biomolecules such as proteins containing multiple amino groups (i.e., lysines), as many amino groups as desired can be reacted with the linkers. The degree of modification can be controlled by the number of molar equivalents of the linking compounds used. In other embodiments, an antigen may have one or more terminal thiol groups (eg, typically multiple) from cysteine ​​residues that may be reactive, eg, with an E group (eg, the maleimide or disulfide group) on the linker. Again, the degree of modification can be controlled by the number of molar equivalents of the linking compounds used.

The reaction of an antigen and a linker can be carried out in an appropriate buffered solution (for example, in a phosphate buffer at a pH in the range of 7.2 to 7.5). The linker can be dissolved in an appropriate polar solvent (eg, DMSO or DMF) and combined with the antigen-containing buffer. The reaction can conveniently be carried out at room temperature. In some embodiments, the linker is a compound represented by

<img file="MX347240B_D0068.tif" />

and C (O) -NH- (CH₂CH₂OR)_p-CH₂CH₂-C (O) -LO A— / where A, p, and LG are as defined above in any of its modalities, and the modified antigen has at least one segment represented by the formula:

A— '* where A and p are as defined above in either embodiment, and the nitrogen atom indicated by N * is covalently bound to the antigen.

In some embodiments of making a conjugate according to the present invention, the hydrazine or hydrazide-substituted immune response modifier can be dissolved in an appropriate polar solvent (eg, DMSO, DMF) and combined with an appropriate buffer solution. of the linker modified antigen as described above. In some embodiments, the hydrazine or hydrazide functional group can be protected with an acid-labile protected amino group, for example, by forming an imine (for example, isopropylidenehydrazino group as shown in Formulas XV and XVII in Reaction Scheme II above. ) or a carbamate (e.g. ter62

IMPI

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INSTITUTO MEXICANO at la ntoniDAS

INDUSTRIAL butoxycarbonylamino). In these embodiments, an acidic buffer (for example, with a pH in the range of

4.7 to 6.2) can effect deprotection of the amino group and allow reaction with antigen possessing aldehyde at the same time. The reaction is typically carried out at room temperature. Consequently, in some embodiments of the method of making a conjugate according to the present invention, the method comprises combining an immune response modifier substituted with protected hydrazine or with protected hydrazide; an aldehyde modified antigen as described in any of the above embodiments, and a carrier under conditions where the protected amino group is deprotected and the conjugate is formed.

When a hydrazine- or hydrazide-substituted aromatic immune response modifier is reacted with an antigen modified by the linkers described herein, the reaction of the aromatic aldehyde group with the aromatic hydrazine or hydrazide can be conveniently followed using an assay. UV spectrophotometric. The bis-aromatic hydrazone bond that is formed provides a distinctive chromophore with a maximum absorbance at 354 nm and a molar extinction coefficient equal to 29,000. The number of moles of an MRI compound incorporated into an antigen can be calculated by

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MEXICAN INSTITUTE

OF THE PROPERTY

MDUSTEIAL ^ * &lt;™ Σ ^ ** Divide the conjugate's measured absorbance at 354 nm by the molar extinction coefficient of 29,000 as demonstrated in the Examples below.

To promote solubility and stability in the reaction, to provide the conjugates described herein, various additives in the reaction mixture may be useful, depending on the properties of the antigen or the selected protein. For example, glycerol and / or surfactants (eg, polysorbate 80) can be useful to promote solubility and stability. Conveniently, since linkers according to and / or useful for practicing the present invention include a poly (ethylene glycol) segment, they promote the solubility and stability of a protein without the addition of glycerol and / or surfactants. To promote reaction efficiency, catalysts (eg, aniline) can be added in effective amounts (eg, up to 200 mM). Advantageously, however, the hydrazone linkages in the conjugates according to the present invention can be made without catalysis.

Conjugates of the invention can also be prepared using the synthetic routes described in the EXAMPLES below.

In some embodiments, the antigen is a protein. Exemplary proteins that may be useful

<img file="MX347240B_D0070.tif" />

antigens in conjugates of the

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MEXICAN INSTITUTE OF PROHIDAL¹INDUSTRIAL invention include hemagglutinin from H1N1 PR8 ^ -901-099100 (009000 ^ 001-1 ^ 4 .-) 1.-1 (a rr »TJr * Hepatitis B surface antigen, Leishmania antigen, secretory protein

Respiratory syncytial virus F, malaria surface antigen, prostate alkaline phosphatase prostate cancer antigen, and M phase phosphoprotein 1 bladder cancer antigen.

Optimal reaction conditions may vary according to variant characteristics of the protein, including isoelectric point, large average hydropathy, instability index (an estimate of the stability of the protein in a test tube), volume relative occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine), which is considered a positive factor for increasing the thermostability of globular proteins, the number of anionic residues, and the number of cationic residues. Such characteristics are known for a variety of proteins.

The stability of proteins and the maintenance of their native conformations are subjected to a combination of hydrophobic interactions within their internal domains and hydrogen bonding and charge interactions on the outer surface of their structure. Since these surface interactions are altered by the modification⁶⁵ ΙΜΡΙ ^ ξ MIXICANC institute

SAY THE PROPERTY ISmSLÍ

INDUSTRIAL "with reagents such as linkers according to and / or using to practice the present invention, the native conformation of the protein can be altered. To provide the conjugate (ie, the MRI reaction product, the linker, and a protein), a ratio of the linker to the protein can be varied, such that the stability of the protein and its native conformation are maintained. In some embodiments, a ratio of linker to protein is in a range of 30: 1 to 1: 3. In some embodiments, a ratio of linker to protein is in a range of 20: 1 to 1: 2. In some embodiments, a ratio of linker to protein is in the range of 10: 1 to 1: 1. The number of equivalents of the immune response modifier can be the same or similar to the number of equivalents of the linker used in some embodiments. In some embodiments, a ratio of the conjugated MRI to the protein is in a range of 30: 1 to 1: 6. In some embodiments, a ratio of the MRI conjugated to the protein is in a range of 20: 1 to 1: 5. In some embodiments, a ratio of the MRI conjugated to the protein is in a range of 10: 1 to 1: 1.

As shown in the EXAMPLES below, conjugates prepared from a linker described herein provide a greater amount of total conjugated protein, a higher amount of conjugated protein

<img file="MX347240B_D0071.tif" />

IMPI

INSTITUTO MEXICANO DE LA NONIDAD INDUSTXIAL soluble, and a higher percentage yield of the conjugated protein »-« μμ »ι I * liJb * -.— than the conjugates prepared from a conventional heterobifunctional linker: succinimidyl 4-formylbenzoate (SFB ).

Pharmaceutical Compositions and Methods

A conjugate of the present invention can be administered in a pharmaceutical composition described herein in any suitable manner (eg, non-parenterally or parenterally). As used herein, non-parenterally refers to administration through the digestive tract, including by oral ingestion. Parenterally refers to administration other than through the digestive tract, which could include nasal (for example, transmucosally by inhalation), topical, ophthalmic, and buccal, but in practice it usually refers to an injection (for example, intravenous, intramuscular, subcutaneous, intratumoral, or transdermal) using, for example, conventional needle injection, injection using a microneedle arrangement, or any other known method of injection.

A conjugate of the present invention can be provided in any pharmaceutical composition suitable for administration to a subject, and can be present in the pharmaceutical composition in any suitable form (e.g., a solution, a suspension, an emulsion, or

IMPI

INSTITUTO MEXICANO DE LA PROPERTY INDUSTRIAL any form of mixture). The pharmaceutical composition can be formulated with any pharmaceutically acceptable excipient, carrier or vehicle. The pharmaceutical composition may further include one or more additives including skin penetration enhancers, colorants, fragrances, flavors, humectants, thickeners, suspending agents, surfactants, and dispersing agents.

In addition to the antigens specifically described above and below, the pharmaceutical compositions and methods of the present disclosure may include additional other active agents, for example, in admixture or administered separately. Such additional agents can include an additional chemotherapeutic agent, cytotoxoid agent, antibody, antiviral agent, cytokine, tumor necrosis factor receptor (TNFR) agonist, or immune response modifier. TNFR agonists that can be co-delivered with a conjugate of the present invention (in some embodiments, the conjugate of Formula II) include CD4 0 receptor agonists, as described in US Patent application. Publication No. 2004/0141950 (Noelle et al.). Other active ingredients for use in combination with an MRI preparation of the present invention include those described in, for example, US Patent Application ⁶⁸ IMPI ^ Mixícano Institute

Say THE PROPERTY

INDUSTRIAL

Publication No. 2003/0139364 (Krieg et al.) ..........

Conjugates according to the present description can induce the production of INF-α and TNF-α in human cells. The ability to induce the production of INF-α and TNF-α indicates that the conjugates of the invention can modulate the immune response in a number of different ways, making them useful in the treatment of a variety of disorders. Other cytokines whose production may be induced by administration of the compounds and conjugates described herein generally include Type I interferons (eg, INF-a), IL-1, IL-6, IL-8, IL- 10, IL-12, MIP-1, MCP-1, and a variety of other cytokines. Among other effects, these and other cytokines inhibit virus production and tumor cell growth, making the conjugates of the present invention useful in treating viral disorders and neoplastic disorders. For example, tumor necrosis factor, interferons, or interleukins have been shown to stimulate a rapid release of certain monocyte / macrophage-derived cytokines, and are also capable of stimulating B cells to secrete antibodies that play an important role in antiviral and antitumor activities.

In addition to the ability to induce cytokine production, the conjugates described herein can

<img file="MX347240B_D0072.tif" />

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INSTITUTO MEXICANO Industrial property affect other aspects of the innate immune response.

For example, the activity of natural killer cells can be stimulated, an effect that may be due to cytokine induction. The MRI activity of the conjugate of the present invention may also include the activation of macrophages, which in turn stimulate nitric oxide secretion and the production of additional cytokines. The MRI activity of the conjugate of the present invention may also include induction of cytokine production by T cells, activation of antigen-specific T cells, and / or activation of dendritic cells. Furthermore, the MRI activity of the conjugate can include proliferation and differentiation of B lymphocytes. The MRI activity of the conjugate can also affect the acquired immune response. For example, MRI activity may include induction of cytokine production of IFN-γ type 1 helper T cells (T_H1) and / or inhibition of T-helper cell type 2 IL-4, IL-5 and / or IL-13 cytokine production (T_H2).

A conjugate prepared from an MRI, a linker described herein, and hemagglutinin 1 (HA) may demonstrate a potent vaccine adjuvant effect with a strong T-biased immune response._H1, indicated by the increased ratio of HA-specific IgG2a to HA-specific IgGl antibody. Such responses are

<img file="MX347240B_D0073.tif" />

IMPI

INSTITUTO MEXICANO Di LA PROPERTY INDUSTRIAL typically accompanied by psi-imnlanion _ Hp. It gives — lo-— T cell interferon gamma production and cell-mediated cytotoxic T cell cell-mediated immunity to HA-expressing cells, as well as other vaccine antigens. Such antigens can be those associated with and intended for the treatment of viral and bacterial infections, as well as various cancers.

Consequently, the invention provides a method of inducing cytokine biosynthesis in an animal, which comprises administering an effective amount of the conjugate according to the present invention (or elaborated according to the present invention) to the animal.

In some embodiments of the conjugate of the present invention, the antigen is a vaccine, and the methods according to the invention include a method of vaccinating an animal, which comprises administering to the animal a conjugate according to and / or made according to the present description. Vaccines include any material administered to elicit a humorally and / or cellularly mediated immune response, such as live or attenuated viral and bacterial immunogens and inactivated viral, tumor-derived, protozoan, organism-derived, fungal, and bacterial immunogens. , toxoids, toxins, polysaccharides, proteins, glycoproteins, peptides, cellular vaccines (for example, using Mexican ΐΝπττυτο cells (dendritic wcmtMD rNOwnuAL), DNA vaccines, proteins — nant-PR ^ glycoproteins, and peptides. Exemplary vaccines include vaccines for cancer, BCG, cholera, plague, typhoid, hepatitis A, B, and C, influenza A and B, parainfluenza, polio, rabies, measles, mumps, rubella, yellow fever, tetanus, diphtheria, influenza b for hemophilus, tuberculosis, meningococcal and pneumococcal vaccines, adenovirus, HIV, chicken pox, cytomegalovirus, dengue, feline leukemia, poultry plague, HSV-1 and HSV-2, swine cholera, Japanese encephalitis, syncytial virus respiratory, rotavirus, papillomavirus, severe acute respiratory syndrome (SARS), anthrax, and yellow fever. See also, for example, the vaccines described in International Publication No. WO 02/24225 (Thomsen et al.).

The methods of the present invention can be performed on any suitable subject. Suitable subjects include animals such as humans, non-human primates, rodents, dogs, cats, horses, pigs, sheep, goats, or cows.

The animal to which the conjugate is administered for induction of cytokine biosynthesis or for vaccination may have a disease (eg, a viral or neoplastic disease), and administration of the compound may provide therapeutic treatment.

Also, the conjugate can be administered to the animal before

<img file="MX347240B_D0074.tif" />

IMPI INSTITUTO MEXICANO DE LA MONEDAD INDUSTRIAL that the animal acquires the disease so that the administration of the conjugate can provide prophylactic treatment. For example, a conjugate can be made from an MRI, a linker, and an HIV antigen and can provide therapeutic and / or prophylactic treatment for HIV. In yet another example, a conjugate can be made from an MRI, a linker, and a tumor associated antigen and can provide therapeutic and / or prophylactic treatment against an antigen associated tumor.

Exemplary conditions that can be treated by administration of an MRI conjugate include:

(a) Viral disorders such as disorders resulting from infection with an adenovirus, a herpesvirus (eg, HSV-I, HSV-II, CMV, or VZV), a poxvirus (eg, an orthopoxvirus such as variola or vaccinia , molluscum contagiosum), a picornavirus (for example, rhinovirus or enterovirus), an orthomyxovirus (for example, influenza virus), a paramyxovirus (for example, parainfluenzavirus, measles virus, mumps virus, and respiratory syncytial virus (RSV, a coronavirus (for example, SARS), a papovavirus (for example, papillomavirus, such as those that cause genital warts, common warts or plantar warts), hepadnavirus (for example, the hepatitis virus B), a

<img file="MX347240B_D0075.tif" />

flaviviruses (for example, hepatitis C virus or .....

dengue), or a retrovirus (eg, a lentivirus such as HIV);

(b) Bacterial diseases such as diseases resulting from infection by bacteria, of, for example, the genera Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma , Pneumococcus, Neisseria, Clostridium, Bacillus, Corinebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, or Bordetella;

(c) other infectious diseases such as chlamydia, fungal diseases (eg, candidiasis, aspergillosis, histoplasmosis, or cryptococcal meningitis), or parasite diseases (eg, malaria, pneumocystus carnii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosome infection);

(d) Neoplastic disorders, such as intraepithelial neoplasms, cervical dysplasia, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, Kaposi's sarcoma, melanoma, leukemias (eg, myelogenic leukemia, chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma,

IMPIAS

INSTITUTO MEMCANO yii'Mfeoi DE LA PROPERTY cutaneous T-cell lymphoma, B-keTuT lymphoma, and hairy cell leukemia), breast cancer, lung cancer, prostate cancer, colon cancer, and other cancers;

(e) T-mediated atopic disorders_H2 such as atopic dermatitis or eczema, eosinophilia, asthma, allergy, allergic rhinitis, and Ommen syndrome;

(f) certain autoimmune disorders such as systemic lupus erythematosus, essential thrombocytopenia, multiple sclerosis, discoid lupus, and alopecia areata; and (g) disorders associated with wound repair, such as inhibition of keloid formation and other types of wound healing (eg, enhancement of wound healing, including chronic wounds).

MRI conjugates can also be useful for individuals who have compromised immune function. For example, certain conjugates may be useful for treating opportunistic infections and tumors that appear after suppression of cell-mediated immunity in, for example, transplant patients, cancer patients, and HIV patients.

It will be understood that in the treatment of the disorders mentioned above, for example, the conjugate described herein can be used in combination with other therapies such as the active agents.

IMPI ^ Ksmvro MEXICAN

Μ THE VK »™? ®4DUSTRIAL RWOMEDIA mentioned above and other procedures (eg, chemoablation, laser ablation, cryotherapy, and surgical removal).

An amount of a conjugate effective to induce cytokine biosynthesis is an amount sufficient to cause one or more cell types, such as monocytes, macrophages, dendritic cells, and B cells to produce an amount of one or more cytokines such as, for example , IFN-a, TNF-a, IL-1, IL-6, IL-10 and IL-12 that is increased at an antecedent level of such cytokines. The precise amount will vary according to factors known in the art, but is expected to be a dose of approximately 100 nanograms per kilogram (ng / kg) to approximately 50 milligrams per kilogram (mg / kg), in some embodiments approximately 10 micrograms per kilogram μg / kg) to approximately 5 mg / kg, approximately 100 micrograms / kg to approximately 1 mg / kg, or approximately 0.01 mg / m² up to about 10 mg / m². Alternatively, the dose can be calculated using the actual body weight obtained just prior to the start of a course of treatment. For doses calculated in this way, body surface area (m²) is calculated before the start of the treatment course using the Dubois method: m² = (weight in kg⁰’⁴²⁵ x height cm⁰'⁷²⁵) x 0.007184. An effective amount to treat or

IMPI ^

MEXICAN INSTITUTE

DE LA PMHtDAD VV ^ SaLJ industíial inhibit a viral infection, for example, it is an amount that will cause a reduction in one or more of the manifestations of the viral infection, such as viral lesions, viral load, rate of virus production, and mortality compared to untreated control animals and can include any of the aforementioned doses. An amount of a compound or pharmaceutical composition effective to treat a neoplastic condition is an amount that will cause a reduction in the size of the tumor or in the number of tumor foci, and can include any of the aforementioned doses.

The composition of a formulation suitable for practicing the invention, the precise amount of an effective conjugate for the methods according to the present invention, and the dosage regimen, for example, will vary according to factors known in the art, including nature. of the carrier, the status of the subject's immune system (e.g., suppressed, compromised, stimulated), the method of administering the conjugate, and the species to which the formulation is being administered. Consequently, it is not practical to generally describe the composition of a formulation that includes a conjugate made from a compound of Formula I or IA, an amount of the conjugate that constitutes an effective amount, or a dosage regimen that is effective for all the

<img file="MX347240B_D0076.tif" />

IMPÍ

INSTITUTO MEXICANO DE LA MOHEDAL 'INDUSTRIAL possible techniques, applications.

However,

Appropriate formulations, conjugate amounts, and dosage regimen can readily be determined by those skilled in the art with due consideration of such factors.

In some embodiments, the methods of the present invention include administering a conjugate to a subject in a formulation, for example, having a compound concentration of from about 0.0001% to about 20% (unless otherwise indicated, all percentages provided herein are weight / weight relative to the total formulation), although in some embodiments the conjugate can be administered using a formulation that provides the compound in a concentration outside of this range. In some embodiments, the method includes administering to a subject a formulation that includes from about 0.01% to about 1% of the conjugate, eg, a formulation that includes about 0.1% to about 0.5% compound of the conjugate.

In some embodiments of the methods described herein, the conjugate can be administered, for example, from a single dose to multiple doses per week, although in some embodiments the methods of the present invention can be performed by the ⁷⁸ IMPI ^

IIWHTOTO MEXICAN V ^ fessGYes

OF THE N0RKDAD /%.¾¾

INDUSTRIAL administration of the conjugate at a frequency outside this range. In some embodiments the conjugate can be administered from about once a month to about five times a week. In some modalities the conjugate is administered once a week.

The conjugate can also be used as a booster after initial immunization with a DNA or RNA vaccine that encodes, in whole or in part, the same antigen.

Some Modalities of the Invention:

In a first embodiment, the present invention provides a conjugate comprising a reaction product of:

a hydrazine or hydrazide substituted immune response modifier;

a linker represented by the formula:

<img file="MX347240B_D0077.tif" />

* where A is CH or N, p is in a range of 1 to 50, R is a bond or -alkylene-O-, R 'is alkylene that is optionally interrupted or terminated with one or more amide or ether groups, and E is an amine or thiol reactive group; and an antigen.

In a second embodiment, the present invention

IMPI

INSTITUTO MEXICANO DE LA PROPIEDAD provides the conjugate of the first modality?^IMf! ft^lAlí:

<img file="MX347240B_D0078.tif" />

Immune response modifier s uy L1L III do Ton '' '' ”hydrazine or with hydrazide is substituted with hydrazine and comprises an aromatic ring to which the hydrazine binds.

In a third embodiment, the present invention provides the conjugate of the first or second embodiment, wherein the hydrazine or hydrazide substituted immune response modifier is a hydrazine substituted imidazoquinoline amine, imidazonaphthyridine amine, pyrazoloquinoline amine, pyrazolonaphthyridine -amine, or thiazoloquinolin-amine.

In a fourth embodiment, the present invention provides the conjugate of any of the first to third embodiments, wherein E is selected from the group consisting of maleimide, vinylsulfone, acrylamide, pyridyldisulfide, methylsulfonyl disulfide, Nhydroxysuccinimide ester, sulfo ester -N-hydroxysuccinimide or a salt thereof, 4-nitrophenyl ester, acid chloride, acid bromide, acid anhydride, pentafluorophenyl ester, tetrafluorophenyl ester, Nhydroxybenzotriazole, iodoacetyl, bromoacetyl, chloroacetyl, succinimidyl carbonate, chloroformate, -OC (O) -OCH (C1) CC1 ester₃, -OC (O) -O- (4-nitrophenyl), isocyanate, and thioisocyanate.

In a fifth embodiment, the present invention

<img file="MX347240B_D0079.tif" />

IMPI

ΙΗΓΠπΠΌ MUiCANO PC LA PtOmDAD IWBUmiAL provides the conjugate of the fourth modality, where R 'is alkylene having up to four carbon atoms, and E is an ester selected from the group consisting of N-hydroxysuccinimide ester, sulfo-N- ester hydroxysuccinimide or a salt thereof, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, and Nhydroxybenzotriazole ester.

In a sixth embodiment, the present invention provides the conjugate of any one of the first to fifth embodiments, wherein the antigen is a protein.

In a seventh embodiment, the present invention provides the conjugate of the sixth embodiment, wherein a ratio of the linker to the protein is in a range of 30: 1 to 1: 3.

In an eighth embodiment, the present invention provides the conjugate of any one of the first to fifth embodiments, wherein the antigen is a lipid.

In a ninth embodiment, the present invention provides the conjugate of any one of the first to eighth modalities, wherein the hydrazine or hydrazide substituted immune response modifier is an imidazoquinoline amine, imidazonaphthyridine amine, pyrazoloquinoline amine, or pyrazolonaphthyridine. -amine, each of which is substituted at position 1. In a tenth embodiment, the present invention

<img file="MX347240B_D0080.tif" />

IMPI Mexican iwjrrruTo DE LA MOniDAD INDUSTRIAL provides a conjugate that comprises:

an immune response modifier;

a linker represented by the formula:

<img file="MX347240B_D0081.tif" />

where A is CH or N, p is in a range from 1 to

50, R is a bond or -alkylene-O-, and R 'is a bond or alkylene that is optionally interrupted or terminated with one or more amide or ether groups; and an antigen, wherein the immune response modifier is covalently linked to the linker at * through a hydrazone functional group, and wherein the antigen is covalently linked to the linker at ** through an amide, disulfide, urea, thiourea, carbamate, or a carbon-sulfur or carbon-nitrogen bond, alpha position to an amide or sulfone, or directly linked to a succinimide ring. In some of these embodiments, the antigen is covalently linked to the linker at ** through an amide functional group.

In a eleventh embodiment, the present invention provides the conjugate of the tenth embodiment, wherein the immune response modifier is an imidazoquinoline amine, imidazonaphthyridine amine,

IMPI ^

Mexican ΓΜτττυτο

M THE PROPERTY

INDUSTRIAL í pyrazoloquinolin-amine, pyrazolonaphthyridine-amine, oa ^ ®ΜΡββ ·· ΐ ^ ··· ι · 1Β ······ ®Γ5 ··; ± ϋΡΡί3ϊ-ί ».ί®ρ · τ · τ · ^ &lt;thiazoloquinolin-amine.

In a twelfth embodiment, the present invention provides the conjugate of the eleventh embodiment, wherein the immune response modifier is an imidazoquinoline amine, imidazonaphthyridinamine, pyrazoloquinoline amine, or pyrazolonaphthyridine amine, and wherein the hydrazone functional group it is located at the 1-position of imidazoquinoline-amine, imidazonaphthyridinamine, pyrazoloquinoline-amine, or pyrazolonaphthyridine-amine.

In a thirteenth embodiment, the present invention provides the conjugate of any one of the tenth to twelfth embodiments, wherein the antigen is a protein.

In a fourteenth embodiment, the present invention provides the conjugate of any of the tenth to twelfth embodiments, wherein the antigen is a lipid.

In a fifteenth embodiment, the present invention provides the conjugate of any one of the tenth to fourteenth embodiments, wherein the hydrazone functional group is linked to an aromatic ring of the immune response modifier.

In a sixteenth embodiment, the present invention provides the conjugate of any of the

IMPI ^

INJTHWTP MEXICANO Bí LA FROHEDAD industrial first to fifteenth modalities, where A is CH.

nwmrr. i - i --- In a seventeenth embodiment, the present invention provides the conjugate of any of the first to fifteenth embodiments, where p is in a range of 2 to 16.

In an eighteenth embodiment, the present invention provides a method of making the conjugate of any of the first to seventeenth embodiments, the method comprising:

combining an antigen with a linker to provide a modified antigen, where the linker is represented by the formula:

0^{Z /}* ff CIOtNH-R'XCHjCJ ^ O) -R'-EA— where A is CH or N, p is in a range of 1 to 50, R is a bond or -alkylene-O-, R 'is a bond or alkylene © which is optionally interrupted or terminated with one or more amide or ether groups, and E is an amine or thiol reactive group; and combining the modified antigen with a hydrazine or hydrazide substituted immune response modifier to provide the conjugate.

In a nineteenth embodiment, the present invention provides a method of making a

<img file="MX347240B_D0082.tif" />

IMPI

ΙΝΓΠΤυΤΟ MEXICAN

OF THE FEOPTIDAr

INDUSTRIAL conjugate, the method comprising:

combining an antigen with a linker to provide a modified antigen, where the linker is represented by the formula:

<img file="MX347240B_D0083.tif" />

* where A is CH or N, p is in a range of 1 to 50, R is a bond or -alkylene-O-, R 'is a bond or alkylene that is optionally interrupted or terminated with one or more amide groups or ether, and E is an amine or thiol reactive group; and combining the modified antigen with a hydrazine or hydrazide substituted immune response modifier to provide the conjugate.

In a twentieth embodiment, the present invention provides the method of the nineteenth embodiment, wherein the hydrazine- or hydrazide-substituted immune response modifier is hydrazine-substituted, and comprises an aromatic ring to which the hydrazine binds.

In a twenty-first embodiment, the present invention provides the method of the nineteenth or twentieth embodiment, wherein the hydrazine-substituted or hydrazide-substituted immune response modifier is a hydrazine-substituted imidazoquinoline amine,

IMPI ^

MEXICAN INSTITUTE

Say THE industrial PROPERTY imidazonaphthyridine-amine, pyrazoloquinoline-amine, pyrazolonaphthyridine-amine, or a thiazoloquinoline-amine.

In a twenty-second embodiment, the present invention provides the method of any of the nineteenth to twenty-first embodiments, wherein the hydrazine- or hydrazide-substituted immune response modifier is an imidazoquinoline amine, imidazonaphthyridine amine, pyrazoloquinoline- amine, or pyrazolonaphthyridine-amine, each of which is substituted at the 1-position.

In a twenty-third embodiment, the present invention provides the method of any of the nineteenth to twenty-second embodiments, wherein E is selected from the group consisting of maleimide, vinylsulfone, acrylamide, pyridyldisulfide, methylsulfonyl-disulfide, N-hydroxysuccinimide ester , sulfo-N-hydroxysuccinimide ester or a salt thereof, 4-nitrophenyl ester, acid chloride, acid bromide, acid anhydride, pentafluorophenyl ester, Tetrafluorophenyl ester, Nhydroxybenzotriazole, iodoacetyl, bromoacetyl, chloroacetyl, succinimidyl carbonate, chloroformate, -OC (O) -OCH (C1) CC1₃, -OC (O) -0- (4-nitrophenyl), isocyanate, and thioisocyanate.

In a twenty-fourth embodiment, the present invention provides the method of the twenty-third

IMPIOUS

MEXICAN INSTITUTE

FROM INDUSTRIAL PROPERTY ^ ¿¿2 = modality, where R 'is alkylene having up to four carbon atoms, and E is an ester selected from the group consisting of N-hydroxysuccinimide ester, sulfo-Nhydroxysuccinimide ester, or a salt thereof, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, and N-hydroxybenzotriazole ester.

In a twenty-fifth embodiment, the present invention provides the method of any one of the nineteenth to twenty-fourth embodiments, wherein the antigen is a protein.

In a twenty-sixth embodiment, the present invention provides the method of the twenty-fifth embodiment, wherein a ratio of the linker to the protein is in a range of 30: 1 to 1: 3.

In a twenty-seventh embodiment, the present invention provides the method of any one of the nineteenth to twenty-fourth embodiments, wherein the antigen is a lipid.

In a twenty-eighth embodiment, the present invention provides the method of any one of the nineteenth to twenty-seventh embodiments, wherein A is CH.

In a twenty-ninth embodiment, the present invention provides the conjugate of any one of the nineteenth to twenty-eighth embodiments, wherein p is in a range of 2 to 16.

<img file="MX347240B_D0084.tif" />

In a thirtieth embodiment, the present invention

IMPI

INSTITUTO Mexicano M LA nteniDAD INDUSTRIAL provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the conjugate of any of the first to seventeenth modalities.

In a thirty-first embodiment, the present invention provides a method of vaccinating an animal, the method comprising administering an effective amount of the conjugate of any one of the first to seventeenth modalities or the pharmaceutical composition of the thirty-seventh embodiment to the animal.

In a thirty-second embodiment, the present invention provides a method of inducing cytokine biosynthesis in an animal, the method comprising administering to it an effective amount of the conjugate of any of the first to seventeenth modalities or the pharmaceutical composition of the thirty-seventh modality to the animal.

In a thirty-third embodiment, the present invention provides a conjugate or pharmaceutical composition for use in vaccinating an animal by administering an effective amount of the conjugate of any one of the first to seventeenth modalities or the pharmaceutical composition of the thirty-seventh embodiment to the animal. .

In a thirty-fourth embodiment, the present invention provides a conjugate or pharmaceutical composition

<img file="MX347240B_D0085.tif" />

IMPI

MEXICAN INSTITUTE

OF THE INDUSTRIAL WOHEDAD for use in stimulating a specific antigen response in an animal, by administering an effective amount of the conjugate of any of the first to seventeenth modalities or the pharmaceutical composition of the thirty-seventh modality to the animal.

In a thirty-fifth embodiment, the present invention provides a conjugate or pharmaceutical composition for use in inducing cytokine biosynthesis in an animal by administering an effective amount of the conjugate of any one of the first to seventeenth embodiments or the composition. Pharmaceutical of the thirtieth modality to the animal.

In a thirty-sixth embodiment the present invention provides a compound represented by the formula:

<img file="MX347240B_D0086.tif" />

"Where A is CH or N, p is in a range of 1 to 50, and LG is a group that can be displaced by an amine.

In a thirty-seventh embodiment, the present invention provides the compound of the thirty-sixth embodiment, wherein LG is selected from the group consisting of N-succinimidyloxy, p-nitrophenoxy, pentafluorophenoxy, N89

<img file="MX347240B_D0087.tif" />

IMPI

MUICANO INSTITUTE

OF THE INBUSTaiAL impiety benzotriazolyloxy, and sodium thereof.

sulfo-N-succinimidyloxy or a salt of

In a thirty-eighth embodiment, the present invention provides the compound of the thirty-sixth or thirty-seventh embodiment, wherein p is in a range of 2 to 16.

In a thirty-ninth embodiment, the present invention provides a modified antigen having at least one segment represented by the formula:

C (O) -NH- (CH₂CH₂OR)_p-CH₂CH₂-C (O) -N * (H) A— / where A is CH or N, p is in a range of 1 to 50, and the nitrogen atom indicated by N * is covalently bound to the antigen.

In a fortieth embodiment, the present invention provides the modified antigen of the thirty-ninth embodiment, wherein p is in a range of 2 to 16.

In a forty-first embodiment, the present invention provides the modified antigen of the thirty-ninth or fortieth embodiment, wherein the antigen is a protein.

In a forty-second embodiment, the present invention provides the conjugate or method of any of the first to twenty-ninth embodiments except the ⁹⁰ IMPI ^

MEXICAN INSTITUTE

M THE nOPIETY Vj ^ an ^^ - s

INBUSTtIAL modalities 9, 12, and 22, where the immune response modifier is an imidazoquinoline amine, imidazonaphthyridine amine, pyrazoloquinoline amine, pyrazolonaphthyridine amine, or a thiazoloquinoline amine, each of which is conjugated through from position 7.

The embodiments of this invention are further illustrated by the following non-limiting examples, but the particular materials and amounts thereof indicated in these examples, as well as other conditions and details, should not be construed as unduly limiting of this invention.

EXAMPLES

Example 1

Compound A

<img file="MX347240B_D0088.tif" />

Part A

CA (PEG) 12 (formula of H₂N-CH₂CH₂- (OCH₂CH₂) i₂-CO₂H;

Molecular Weight (MW) = 617.7; obtained from Thermo Scientific, Rockford, IL, 115 mg) dissolved in anhydrous dichloromethane (5 ml), N-succinimidyl-4-formylbenzoate (52 mg dissolved in anhydrous dichloromethane (0.5 ml) obtained from EMD Chemicals, Gibbstown, NJ), triethylamine anhydrous (52 pL), and a catalytic amount of DMAP were combined under an atmosphere of

<img file="MX347240B_D0089.tif" />

nitrogen. The reaction was stirred for 3 hours and then diluted with dichloromethane (25 ml). The organic fraction was washed with 0.1 M sodium phosphate (2 x 10 ml) followed by brine. The organic fraction was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The aqueous wash fractions were combined and extracted with several portions of dichloromethane. The aqueous fraction was then acidified to pH ~ 2 with dilute hydrochloric acid and extracted with two additional portions of dichloromethane. The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting material was combined with the material obtained from the first extraction and purified using a small column of silica gel. Elution with 10-25% methanol / chloroform saturated with water produced 58 mg of the amide product as a colorless solid. ¹H NMR (chloroform-d, 500 MHz) δ 10.08 (s, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H), 7.19 (m, 1H), 3.77 (t, J = 6.1 Hz, 2H), 3.70-3.60 (m, 48H), 2.60 (t, J = 6.1 Hz, 2H).

Part b

The material from Part A was dissolved in anhydrous N, N-dimethylformamide (0.5 ml) and anhydrous pyridine (0.5 ml). 0- (N-succinimidyl) 1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU; 46 mg; available from Sigma Aldrich, St. Louis, MO) was added and the reaction stirred under a

<img file="MX347240B_D0090.tif" />

⁹² IMPI

MEXICAN INSTITUTE

OF THE reOriEDAP

INDUSTRIAL nitrogen atmosphere for 3 hours. Most of the solvent was removed under reduced pressure. The resulting material was dissolved in chloroform (25 ml) and methanol (5 ml) and placed in a separatory funnel. A buffer solution (10 ml of a 0.10 M sodium chloride solution, 0.05 M sodium phosphate, 1.0 mM EDTA adjusted to pH 7.5 with sodium hydroxide) was added and the mixture was stirred for 2 minutes. The organic fraction was collected and washed sequentially with an additional portion of the buffer (10 ml), water (3 x 10 ml), and brine. The organic fraction was dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide 55 mg of compound A as a colorless syrup.^XH NMR (chloroform-d, 500 MHz) δ 10.08 (s, 1H), 7.99 (d, J = 8.2 Hz, 2H), 7.95 (d, J = 8.1 Hz, 2H), 7.10 (τη, 1H), 3.85 (t, J = 6.5 Hz, 2H), 3.70-3.60 (m, 48H), 2.90 (t, J = 6.9 Hz, 2H) 2.84 (s broad, 4H).

Example 2

Recombinant hemagglutinin 1 (HA) from H1N1 PR8 was cloned, expressed in E. coli, γ purified using standard procedures. The HA, with a molecular weight of 32083.11 Daltons, which has 6 histidines at the C end, was placed in a 0.1 M buffer of pH 7.5, containing 0.15 M sodium chloride. Based on the molecular weight of the HA and the mass of protein, it was established

<img file="MX347240B_D0091.tif" />

⁹³ IMPI Mexican institute

M LA RRDFI age

INDUSTRIAL the molarity of the HA solution. The -compound A dissolved in dimethyl sulfoxide (DMSO) was added to the HA at a 10-fold molar excess. The solution was then incubated for 2 hours at room temperature. HA modified with compound A (represented as HA-compound A) was separated from free compound A by using a rotating ZEBA column (Thermo Scientific, Rockford, IL) pre-equilibrated with 0.1 M phosphate buffer pH 6.0, containing 0.15 M sodium chloride. This step changed the HA-Compound A solution to pH 6.0 in preparation for the conjugation reaction.

Example 3

N- (4 - {[4-amino-2-butyl-lH-imidazo [4,5-c] guignolin-1-yl] oxy} butyl) -6- (Ν'-isopropylidenehydrazino) nicotinamide (prepared as described below ) was dissolved in DMSO and added to the HA-Compound A buffer in a 10-fold molar excess. The acidic conditions of the reaction medium resulted in the deprotection of the acetimine protecting group of N- (4- {[4-amino-2-butyl-1Himidazo [4,5-c] quinolin-l-yl] oxy} butyl) -6- (Ν 'isopropylidenehydrazino) nicotinamide to form the N- (4 - {[4 amino-2-butyl- lH-imidazo [4,5-c] quinolin-l-yl] oxy} butyl) -6hydrazinonicotinamide in if you. The sample was incubated for 2 hours at room temperature. HA-Compound A covalently conjugated to N- (4- {[4-amino-2-butyl-lH94

<img file="MX347240B_D0092.tif" />

Imidazo [4,5-c] guignolin-l-yl] oxy} butyl) -6 hydrazinonicotinamide (represented as HA-Compound A Compound 2) was separated from unconjugated components by using a ZEBA rotating column pre-equilibrated with solution Dulbecco's phosphate buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO).

Preparation of

N- (4 - {[4-amino-2-butyl-lH-imidazo [4,5-c] quinolin-1-yl] oxy} butyl) -6- (Ν'-isopropylidenehydrazino) nicotinamide (Compound 1) and N- (4- {[4-amino-2-butyl-lH-imidazo [4,5c] quinolin-l-yl] oxy} butyl) -6-hydrazinonicotinamide (Compound

2)

<img file="MX347240B_D0093.tif" />

(Compound 1) H (Compound 2) H

Part A

A solution of valeric anhydride (6.03 g) and pyridine hydrochloride (0.198 g) in pyridine (8.28 g) was added to a solution of 3-amino-4-chloroquinoline (2.94 g) in pyridine (5.0 g) and the reaction was stirred at room temperature for 16 hours, followed by heating at 60 ° C for 3 hours. The reaction was concentrated under reduced pressure and

<img file="MX347240B_D0094.tif" />

added sodium carbonate (15 ml of a 10% aqueous solution). The reaction was stirred for 30 minutes and then filtered. The resulting solid was washed with water (60 ml) and dried under vacuum for 4 hours to provide 4.59 g of the crude N- (4-chloroquinolin-3-yl) valeramide as brown flakes. The crude product was recrystallized from heptane (10 ml) and the recovered product was further purified by soxhlet extraction using refluxing heptane for 16 hours. The collection flask from the soxhlet extraction apparatus was chilled in a refrigerator for 2 hours. The resulting solid was collected by filtration and dried under vacuum to yield 2.00 g of the N- (4-chloroquinolin-3-yl) valeramide as a white solid. Part b

A solution of 4-amino-l-butanol (7.68 g) and pyridine (7.00 g) in dichloromethane (100 ml) was cooled in an ice bath and a solution of benzyl chloroformate (14.37 g) in dichloromethane ( 100 ml) with agitation over a thirty minute period. The ice bath was removed and the reaction was stirred for an additional 16 hours. Hydrochloric acid (1.2 M, 200 ml) was added and the phases were separated. The organic phase was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was recrystallized from toluene and dried in vacuo to provide 5.15 g of (496

ΙΜΡΙ®5

MEXICAN INSTITUTE

OF THE PROPERTY VJ INDUSTRIAL hydroxybutyl) benzyl carbamate.

A solution of N-hydroxyphthalimide (3.36 g), benzyl (4-hydroxybutyl) carbamate (4.18 g) and triphenylphosphine (7.41 g) in dichloromethane (100 ml) was cooled in an ice bath and approximately two-thirds of an azodicarboxylate solution of Diisopropyl (DIAD, 5.68 g) in dichloromethane (50 ml) was added slowly with stirring. The internal temperature of the reaction was monitored and the addition of the DIAD solution was stopped when an exotherm could no longer be detected. The ice bath was removed and the reaction was allowed to warm to room temperature. The reaction was concentrated under reduced pressure and the resulting residue was dissolved in ethanol (200, 100 ml test). Hydrazine (1.98 g, 35% in water) was added and the reaction was stirred for 6 hours. The reaction was cooled in the refrigerator and the resulting solution was filtered off. The solid was washed with ethanol (50 ml). The combined filtrate was concentrated under reduced pressure and diethyl ether (100 ml) was added. Insoluble impurities were removed by filtration and 2.0 M HC1 in ether (10 ml) was added to the solution. A precipitate formed immediately. The crude product was added to toluene (10 0 ml) and heated at reflux temperature for one hour. After cooling to room temperature, the solid product was recovered by filtration, washed with toluene, and dried under vacuum to yield

<img file="MX347240B_D0095.tif" />

3.76 g of benzyl (4-aminoxybutyl) carbamate.

Part c

N- (4-chloroquinolin-3-yl) valeramide (1.97 g), benzyl (4-aminoxybutyl) carbamate (2.99 g), triethylamine (0.89 g) and 2-propanol (40.69 g) were combined, and heated to 80 ° C for 3.5 hours. The reaction was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. Dichloromethane (20 ml) was added to the resulting solid and the mixture was stirred for twenty minutes. The undissolved solid was removed by filtration and the filtrate was washed with two 10 ml portions of water which had been made slightly acidic by the addition of 20 drops of hydrochloric acid (1.2 M). The organic fraction was dried and concentrated under reduced pressure. The crude solid was recrystallized from tetrahydrofuran to provide 2.56 g of benzyl 4 - {[2-butyl-lH-imidazo [4,5c] quinolin-1-yl] oxy} butylcarbamate. Part d

Benzyl 4 - {[2-butyl-lH-imidazo [4.5c] quinolin-1-yl] oxy} butylcarbamate hydrochloride (10.05 g) was dissolved in dichloromethane (80 ml) and extracted with a carbonate solution sodium (2.02 g) in 30 ml of water. The organic layer was cooled in an ice bath and a solution of m-chloroperbenzoic acid (5.93 g,

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IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

1.24 eq) dissolved in dichloromethane (30 ml). After 6 hours, ammonium hydroxide (10 ml of a 28-30% aqueous solution) was added to the reaction. A solution of benzenesulfonyl chloride (6.96 g) dissolved in 10 ml of dichloromethane was added slowly with vigorous stirring. The cooling bath was removed and the reaction was stirred for an additional 12 hours. The reaction was diluted with water (100 ml) and the organic and aqueous fractions were separated. The aqueous fraction was extracted with dichloromethane (30 ml). The combined organic fractions were washed with two 90 ml portions of 5% sodium carbonate.

The dichloromethane solution was transferred to a distillation apparatus and 1-pentanol (50 ml) was added. This was heated to 40 ° C and the dichloromethane was removed under reduced pressure. Then concentrated hydrochloric acid (50 ml) was added and the reaction was stirred and heated to 80 ° C. After 11 hours, the solution was cooled to room temperature and diluted with water (100 ml). The aqueous fraction was separated from the 1-pentanol and the 1-pentanol was extracted with water (25 ml). The aqueous fractions were combined. 1-Pentanol (50 ml) was added to the combined aqueous fraction, and this was cooled in an ice bath. With vigorous stirring, solid sodium carbonate was added to bring the pH to 9-10. The mixture was transferred to a separatory funnel and the fractions were separated. The aqueous fraction

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⁹⁹ IMPI

ΙΗΓΓΠνΤΟ MEXICANO DE LA MOMEDAD INDUSTRIAL was extracted with two 25-ml portions of 1-pentanol. The combined 1-pentanol fractions were dried over sodium sulfate and filtered to provide the l- (4-aminobutoxy) -2-butyl-lH-imidazo [4,5-c] quinolin-4-amine dissolved in 1-pentanol.

The maleate salt of 1- (4-aminobutoxy) -2-butylIH-imidazo [4,5-c] quinolin-4-amine was prepared by dissolving maleic acid (4.83 g) in 1-pentanol (50 ml ) and adding it with stirring to the solution of 1- (4-aminobutoxy) 2-butyl-lH-imidazo [4,5-c] quinolin-4-amine in 1-pentanol. The resulting precipitate was collected by filtration and dried to yield 7.69 g of 1- (4-aminobutoxy) -2-butyl-lHimidazo [4,5-c] quinolin-4-amine as the bis maleate salt.^XH NMR (DMSO-d6): δ 0.96 (t, 3H), 1.44 (m, 2H), 1.7-1.95 (m, 4H), 2.02 (m, 2H), 2.8-3.1 (m, 4H), δ 4.43 (t, 2H), 6.07 (s, 4H), 7.57 (t, 1H), 7.73 (t, 1H), 7.80 (d, 1H), 8.16 (d, 1H). Wide peaks for the ammonium protons are observed at approximately δ 7.8 and δ 8.7. Part e

The bis-maleate salt of 1- (4-aminobutoxy) -2-butylIH-imidazo [4,5-c] quinolin-4-amine (0.2 g) was suspended in 1-butanol (5 ml) and washed sequentially with 2 5 ml portions of a 5% sodium carbonate solution, followed by 5 ml of a saturated sodium chloride solution. Acetone hydrazone of succinimidyl 4-hydrazinonicotinate (SANH,

100 IMPI

MEXICAN INSTITUTE

OF THE PROPERTY Í&gt; a-.¿S

INDUSTRIAL ^ a. 0.0216 g); available from Thermo Scientific, Ruekfuid,¹ IL, wasadded and the solution was stirred at room temperature for 17.5 hours. Analysis of the reaction by thin layer chromatography (silica gel, eluent of methyl tert-butyl ether: ethanol 1: 1) showed only the presence of 1 (4-aminobutoxy) -2-butyl-1H-imidazo [4 , 5-c] guignolin-4-amine (R_F&lt;0.05) and the desired product (N- (4- {[4-amino-2-butyl-lHimidazo [4,5-c] quinolin-1-yl] oxy} butyl) -6- (Ν 'isopropylidenehydrazino) nicotinamide (Rf 0.30). The reaction was concentrated under reduced pressure and 5 ml of dichloromethane was added to the residue. Small amounts of insoluble material were removed by filtration and the sample was purified by column chromatography (silica gel, eluent tert-butyl ether methyl: ethanol 1: 1). The fractions containing the product were combined and the solvent removed under reduced pressure to provide the N- (4 - {[4-amino-2-butyl-lH-imidazo [4,5-c] quinolin-1-yl] oxy} butyl ) -6- (Ν '-isopropylidenehydrazino) nicotinamide as a light yellow solid (compound 1).

NMR (chloroform-d) δ: 8.59 (d, J = 2.2 Hz, 1H), 7.81-8.15 (m, 3H), 7.75 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 7.20 (d, J = 8.7 Hz, 1H), 6.57 (t, J = 5.6 Hz, 1H), 5.61 (broad s, 2H), 4.24 (t , J = 6.1 Hz, 2H), 3.55 (q, J = 6.3 Hz, 2H), 2.88 (t, J = 7.6 Hz, 2H), 1.93-2.12 (m, 5H), 1.74-1.93 (m, 7H) , 1.37-1.54 (m, 2H),

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N- (4- {[4-amino-2-butyl-lH-imidazo [4,5IMPI Mexican institute DI LA PROPIEDAD INDUSTRIAL

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0.96 (t, J = 7.2 Hz, 3H).

Part f

The

c] quinolin-1-yl] oxy} butyl) -6- (Ν 'isopropylidenehydrazino) nicotinamide from Part E was suspended in 1 ml of hydrochloric acid (0.6 M) and heated to

60 ° C for 90 minutes. The resulting homogeneous solution was cooled to room temperature and the reaction was concentrated under reduced pressure. The resulting residue was dissolved in water and lyophilized to provide 43.6 mg of the hydrochloride salt of N- (4- {[4-amino-2-butyl-lHimidazo [4,5-c] quinolin-l-yl] oxybutyl) -6hydrazinonicotinamide as a yellow solid (Compound 2). MS (ESI) m / z 463.25661 (463.25645 calculated for C₂₄H₃₁N₈OR₂, M + H₊) .

Comparative Example A

Comparative Example A was prepared according to the methods of Examples 2 and 3, with the modification that succinimidyl 4-formylbenzoate (SFB) (Thermo

Scientific, Rockford, IL) dissolved in dimethyl sulfoxide (DMSO) instead of compound A, was added to HA at a 10-fold molar excess during the Example 2 step.

The efficiency of incorporation of Compound 2 into HA through covalent conjugation was determined using a spectrophotometric assay.

102

IMPI ^ ¡mirtvro Mexican

DE U MIOPIEDAP k.&gt; - ÍS INDUSTRIAL - de UV. This bis-aromatic hydrazone bond that is formed by the covalent conjugation of HA-SFB with Compound 2, provides a distinctive chromophore. Chromophore has a maximum absorbance at 354 nm and a molar extinction coefficient equal to 29,000. The number of moles of Compound 1 incorporated into the HA protein was calculated by dividing the measured absorbance of the HA-SFB-Compound 2 conjugate at 354 nm by the molar extinction coefficient of 29,000. The calculated moles of compound 2 covalently conjugated to one mole of the HA-SFB protein was 6.1.

Comparison of the Conjugation Methods of Example 3 and Comparative Example A

The effect of using Compound A in the covalently conjugated product, compared to SFB, on the final protein solubility and percent recovery, is shown in Table I. The soluble protein measurement was determined as the amount of Comparative Example A or Example 3 recovered in the supernatant of a centrifuged sample of 100K x g. The total protein measurement was determined as the amount of Comparative Example A or Example 3 in the sample before centrifugation. Soluble protein and total protein measurements were performed using a Bicinchoninic Acid (BCA) Protein Assay (obtained from Thermo Scientific, Rockford, IL).

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<img file="MX347240B_D0099.tif" />

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INSTITUTO MEXICANO OE LA FROPIEDAIj INDUSTRIAL

Table I

<td>Sample of Protein</td><td>Total Protein (μ ^ πύ)</td><td>Soluble Protein W^ml)</td><td>Percentage of Recovery</td>

<td>Example Comparative A</td><td> 630.2</td><td> 215.8</td><td> 34.2%</td>

<td>Example 3</td><td> 686.9</td><td> 659.3</td><td> 95.9%</td>

Example 4 - Prophetic

In vitro induction of interferon-α (IFN) and production of tumor necrosis factor (TNF) in human peripheral mononuclear cells (PBMC) by the conjugate of Example 3, can be determined using the following procedure. PBMCs prepared from human volunteers can be cultured in 96-well microtiter plates. HA, the modified HA from Example 2, and the conjugate from Example 3 can be added to the wells at a final concentration of 1 µΜ protein. Cells can be incubated overnight at 37 ° C. The medium can be removed and the IFN concentration (pg / ml) and the TNF concentration (ng / ml) can be measured by ELISA.

Example 5 - Prophetic

The adjuvant activity of the vaccine conjugate from Example 3 can be evaluated in male Balb / C mice.

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IMPI lÓSTlTVÍ © MEXICANO Of La phoHídad Industrial (Charles River

Groups of 5

Laboratories, International, Wilmington, MA).

* 4 'ιΐ'ίιί mice can each be immunized subcutaneously with 10 micrograms of HA antigen in PBS (control), 10 micrograms of Example 2 (control), or the conjugate of Example 3. Mice can be boosted with the same combinations 2 weeks and 4 weeks after initial immunization. Three weeks and again at 12 weeks after the final boost, the mice can be bled and the HA-specific antibody titers determined. This determination can be carried out by serial dilution of serum samples by standard serum ELISA in HA-coated microtiter plates. Antibody data can be presented as the serum dilution that reaches the end point (2X baseline) and is the geometric mean for the 5 mice per group. As an index of the THl deviation from the immune response, HA-specific IgGl and IgG2a subtypes can be measured, in addition to total HA-specific IgG.

The full descriptions of the patents, patent documents, and publications cited herein are incorporated by reference in their entirety as if each were individually incorporated. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from

<img file="MX347240B_D0101.tif" />

105

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INSTITUID MEXICANO gave INDUSTRIAL ownership of the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples described herein, and that such examples and embodiments are presented by way of example only, with the scope of the invention intended to be limited only. by the group of claims described herein as follows.

It is noted that in relation to this date, the best method known to the applicant for putting the aforementioned invention into practice is the one that is clear from the present description of the invention.

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